Viral vector manufacture

ABSTRACT

This present invention relates to a modified mammalian cell in which the genome of the cell is modified to comprise a sequence encoding CP77 under the control of a promoter such that the modified cell line sustains propagation of a poxvirus that is less able or unable to propagate in the unmodified cell.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/AU2014/050330, filed on Nov. 3,2014, which in turn is associated with and claims priority fromAustralian patent application no. 2013904242 filed on Nov. 1, 2013 andAustralian patent application no. 2014900370 filed on Feb. 7, 2014, theentire contents of each of these applications are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 30, 2016, isnamed 052392-0021_SL.txt and is 72,814 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the development of cells and cell linessuitable for propagating and therefore manufacturing poxvirus-basedmedicaments. In particular, the specification relates to recombinantmodified cellular substrates for propagating such poxviruses for themanufacture of therapeutic or prophylactic agents.

BACKGROUND

Bibliographic details of references in the subject specification arelisted at the end of the specification.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

All publications mentioned in this specification are herein incorporatedby reference in their entirety.

The pox virus family comprises two subfamilies, the Chordopoxvirinae andthe Entomopoxvirinae. The Chordopoxvirinae comprises eight generaincluding the Orthopoxviridae comprising species which infect man (forexample, variola virus, the causative agent of smallpox, cowpox virus(which formed the original smallpox vaccine reported by Jenner in 1796),vaccinia virus (used as a second generation smallpox vaccine) andmonkeypox virus), and the Avipoxviridae viruses comprising species thatinfect birds, such as fowlpox and canarypox viruses. In addition totheir use as antigens in smallpox vaccines, there is much interest inthe use of recombinant vaccinia-based viruses and avipox viruses as a“backbone” vectors. As intra-cytoplasmic vectors, the Orthopoxviridaeare able inter alfa to deliver foreign antigens to the host cytoplasmand antigen processing pathways that process antigens to peptides forpresentation on the cell surface. Such vectors expressing foreignantigens are used in the development of vaccines for diseases such asAIDS, tuberculosis, malaria and cancer which have proven difficult totreat by other vaccination strategies.

The Chordopoxvirinae have linear double-stranded DNA genomes ranging insize from 130 kb in parapoxviruses to over 300 kb in avipoxviruses andtheir life cycle in the host is spent entirely in the host cellcytoplasm. The poxviruses operate substantially independently of theirhost cell and host cell molecules, especially for processes involved inearly mRNA synthesis. However, host molecules appear to be used for theinitiation or termination of intermediate and late viral transcription.The poxviruses produce structurally diverse “host range factors” whichspecifically target and manipulate host signaling pathways to permitcellular conditions allowing viral replication. Most poxviruses can bindand infect mammalian cells, but whether or not the subsequent infectionis permissive (able to produce infectious virions) or non-permissive(substantially unable to produce infectious virions) is dependent uponthe specific poxvirus and specific cell type involved. There iscurrently a relatively poor understanding at the molecular level of poxvirus-host interactions, in particular host-range genes, and whichfactors are necessary to modulate the relationship to facilitate bothviral and cellular propagation. For a review of host range genesreference may be made to Werden et al. 2008 incorporated herein in itsentirety.

Observations on strains of vaccinia relevant to their use as small poxvaccines and subsequently as viral vectors, have been published from theearly 1960's through to the present day. Certain strains of vaccinia,including strains employed as small pox vaccines, are able to propagatein human cells and therefore represent health risks, such as thedevelopment of viral encephalitis. With a view to developing a safervaccine, a vaccinia strain from Ankara (referred to as “CVA”) waspassaged more than 500 times in non-human cells. During this process thevaccinia genome changed substantially involving the development of atleast six major deletions compared to the original CVA genome. Themodified virus was less pathogenic in man but still able to engender aprotective immune response. This attenuated vaccinia virus is referredto as MVA (Modified Vaccinia Ankara) and is also categorized by passagenumber, as viruses with different passage numbers were found to begenetically and phenotypically distinct. However, by passage number 515MVA515 was understood to be genetically stable. In the early 1990s, itwas observed that MVA strains, such MVA572, and its derivative, MVA F6were able to express vaccinia proteins and heterologous (recombinant)proteins at high levels in non-permissive cells (in which the virus willnot propagate), enabling the development of MVA as a vector forheterologous molecules of interest, such as those encoding antigens forvaccine or therapy delivery.

More recently, attempts have been made to produce a modified vacciniavirus with the qualities of MVA by introducing the six large knowndeletions of MVA into CVA. Interestingly, this did not result in a viruswith the attenuated qualities of MVA. It was proposed that the absenceof host range genes might be responsible for the observed attenuation,however this has not been substantiated (see for example, Meyer et al.,Journal of General Virology (1991) 72:1031-1038.

The poxviruses constitute a large family of viruses characterized by alarge, linear dsDNA genome, a cytoplasmic site of propagation and acomplex virion morphology. Vaccinia virus is the representative virus ofthis group of virus and the most studied in terms of viralmorphogenesis. Vaccinia virus virions appear as “brick shaped” or“ovoid” membrane-bound particles with a complex internal structurefeaturing a walled, biconcave core flanked by “lateral bodies”. Thevirion assembly pathway involves a fabrication of membrane containingcrescents which develop into immature virions (IVs), and then evolveinto mature virions (MVs). Over 70 specific gene products are containedwithin the vaccinia virus virion, where the effects of mutations in over50 specific genes on vaccinia virus assembly are now described.

Vaccinia virus enter cells by fusion of its surface membranes with theplasma membrane of the host cell, releasing the core (and lateralbodies) into the cytoplasm and activating the virus' transcriptionalprogram. The virion cores contain the full complement of virus-codedenzymes required for synthesis and modification of early mRNA. Earlygenes encode enzymes required for DNA propagation, and thus as earlygene expression peaks, viral DNA propagation ensues in cytoplasmic sitestermed “factories.” Early genes also encode intermediate transcriptionfactors, and intermediate genes, in turn, encode late transcriptionfactors, so that intermediate and late genes are expressed in successionafter the prerequisite initiation of viral DNA propagation. Thus, thefull complement of viral genes are transcribed in a temporal cascade,with the early, intermediate and late classes being distinguished byclass-specific transcriptional promoters and virally encodedtranscription factors. Furthermore, only propagated genomes arecompetent templates for intermediate and late transcription. These twoclasses of genes together encode virion structural proteins, virionenzymes, and assembly factors and are required for assembly of newprogeny virus particles.

Shortly after viral uptake and early expression infection-specificcytoplasmic domains form within the cell that are uniform in density andare sometimes surrounded by endoplasmic reticulum (ER) derived cisternaewhich increase in size with time. These domains represent sites of viralDNA propagation and are often called “viral factories”.

Viral assembly starts with the formation of rigid crescent-shapedstructures (cupules in three dimensions) within the viral factories. Inhigh resolution electron micrographs the outer layer of these crescentshaped structures are composed of regularly spaced projections termed“spicules”. Crescents apparently grow in length while maintaining thesame curvature until they become closed circles (spheres in threedimensions) called immature virions (IV). IV are filled with “viroplasm”material that is uniform in density but discernibly more electron densethan the surrounding factory. As the IVs form uptake of encapsidated DNAalso takes place: these are seen in electron micrographs as electrondense, round or ovoid subdomain within the IVs called a “nucleoid”. IVsthat contain nucleoids of condensed DNA that are often referred to as“IVN.” Maturation of several virion protein precursors via proteolyticcleavage is required for the morphogenesis of IVN to mature virions(MV). The majority of mature virions are found outside factories and mayexist in clusters either at the periphery of a factory or apparentlyseparated by a significant distance from the nearest factory.

Poxvirus virions exist in three infectious forms: mature virions (MV),wrapped virions (WV), and extracellular virions (EV). MV, the simplestform of the virus, are membraned particles containing a biconcave,DNA-containing core flanked by lateral bodies, which fill theconcavities of the core. MV are normally found exclusively inside cellsand are liberated only by cell lysis. WV consist of MV which aresurrounded by two additional lipid bilayers derived from trans-Golgicisternae. WV, whose outer membranes contain characteristic viralproteins, are precursors of EV and are also found within the cell. EVconsist of WV which have been exocytosed via fusion of the outermost WVmembrane with the plasma membrane, leaving an MV wrapped in oneadditional membrane. A fraction of EV are found attached to the cellsurface, while some are found free in the extracellular medium. EV arethought to be important for spread of the virus within an organism.

Removal of essential genes from viruses and complementation in hostcells is know in the art as a general proposed strategy for generatingsafe and effective viral vectors for treatment and therapy. There is aneed for improved attenuated orthopox vectors with enhanced safety,expression and/or immunogenicity and a commensurate need for methods ofpropagating and manufacturing such orthopox vectors safely andeconomically.

A number of mammalian cell lines are used for the manufacture ofrecombinant proteins for research purposes. These include, rabbit,hamster, primate and human derived cell lines such as, withoutlimitation, and as known in the art, HEK293, 293T, 143B, CHO, HeLa, Veroand BHK, HepG2, and 3T3 cells. Notably, however, the GMP manufacture ofthe majority of recombinant therapeutic proteins have been made in CHOcells making this cell line the most well studied and approved cell linefor human therapeutics. CHO cells can grow to very high densities insuspension cultures, and down stream processes for purifying productsare very well developed. Of relevance, is that neither vaccinia, such asvaccinia-COP or vaccinia-WR nor its derivatives such as MVA and NYVACwill propagate in CHO cells.

Expression of viral host range genes from within the pox virus under thecontrol of local viral promoters and pox viral RNA polymerase is knownin the art.

Expression of certain viral host range genes from the genome of amammalian cell in a timely fashion to rescue or permit viral replicationand permit host cell survival has not previously been described.

SUMMARY

The present specification describes in vitro cultured mammalian cells,transformed to express a poxviral host range factor and a method forpropagating viral vectors, comprising culturing such cells.

In a first aspect the present invention provides a modified mammaliancell in which the genome of the cell is modified to comprise a sequenceencoding CP77 under the control of a promoter such that the modifiedcell line sustains propagation of a poxvirus that is less able or unableto propagate in the unmodified cell.

In a second aspect the present invention provides a process forpropagating an orthopoxvirus that does not propagate in CHO cells, theprocess comprising propagating the poxvirus in vitro in a mammalian cellline wherein the cell line is modified to encode and express CP77 underthe control of a promoter.

In various alternative embodiments the modified cells are furthermodified to comprise a sequence encoding D13L under the control of apromoter and/or to comprise a sequence encoding K1L under the control ofa promoter

Reference herein to “CP77”, “K1L” and “D13L” includes functionalorthologs and functional variants.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

The subject invention is not limited to particular procedures or agents,specific formulations of agents and various medical methodologies, assuch may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. Unless defined otherwise, all technical and scientific termsused herein have the same meanings as commonly understood by one ofordinary skill in the art to which this invention belongs.

Any materials and methods similar or equivalent to those describedherein can be used to practice or test the present invention.Practitioners are particularly directed to: Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborPress, Plainsview, N.Y.; Ausubel et al. (1999) Current Protocols inMolecular Biology (Supplement 47), John Wiley & Sons, New York; Murphyet al. (1995) Virus Taxonomy Springer Verlag: 79-87, Mahy Brian W J andKangro O Hillar (Eds): Virology Methods Manual 1996, Academic Press; andDavison A J and Elliott R M (Eds): Molecular Virology, A practicalApproach 1993, IRL Press at Oxford University Press; Perkus et al.,Virology (1990) 179(1):276-86 or definitions and terms of the art andother methods known to the person skilled in the art.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, preferred methods and materials are described. For thepurposes of the present invention, the following terms are definedbelow.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. Thus, use of the term “comprising” and the likeindicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present. In thecontext of attenuated orthopox vectors, the subject vectors are modifiedfor attenuation by comprising deletion of an essential maturation orassembly gene however, further modification such as to vector an antigenor other protein is encompassed.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

As used herein the singular forms “a”, “an” and “the” include pluralaspects unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a single cell, as well as two ormore cells; reference to “an organism” includes one organism, as well astwo or more organism; and so forth. In some embodiments, “an” means “oneor more than one”.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (or).

“Attenuation” or “attenuated” as used herein means a reduction of viralvector virulence. Virulence is defined as the ability of a virus tocause disease in a particular host. A poxviral vector that is unable toproduce infectious viruses may initially infect cells but is unablesubstantially to replicate itself fully or propagate within the host orcause a condition. This is desirable as the vector delivers its proteinor nucleic acid to the host cell cytoplasm, but does not harm thesubject.

By “control element” or “control sequence” is meant nucleic acidsequences (e.g., DNA) necessary for expression of an operably linkedcoding sequence in a particular poxvirus, vector, plasmid or cell.Control sequences that are suitable for eukaryotic cells includetranscriptional control sequences such as promoters, polyadenylationsignals, transcriptional enhancers, translational control sequences suchas translational enhancers and internal ribosome binding sites (IRES),nucleic acid sequences that modulate mRNA stability, as well astargeting sequences that target a product encoded by a transcribedpolynucleotide to an intracellular compartment within a cell or to theextracellular environment.

Where sequences are provided, corresponding sequences are encompassed.By “corresponds to” “corresponding” or “corresponding to” is meant anucleic acid sequence that displays substantial sequence identity to areference nucleic acid sequence (e.g., at least about 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequenceidentity to all or a portion of the reference nucleic acid sequence) oran amino acid sequence that displays substantial sequence similarity oridentity to a reference amino acid sequence (e.g., at least 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequencesimilarity or identity to all or a portion of the reference amino acidsequence).

By “effective amount”, in the context of treating or preventing acondition or for modulating an immune response to a target antigen ororganism is meant the administration of an amount of an agent (e.g., anattenuated orthopox vector as described herein) or compositioncomprising same to an individual in need of such treatment orprophylaxis, either in a single dose or as part of a series, that iseffective for the prevention of incurring a symptom, holding in checksuch symptoms, and/or treating existing symptoms, of that condition orfor modulating the immune response to the target antigen or organism.The effective amount will vary depending upon the health and physicalcondition of the individual to be treated, the taxonomic group ofindividual to be treated, the formulation of the composition, theassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials.

As used herein, the terms “encode,” “encoding” and the like refer to thecapacity of a nucleic acid to provide for another nucleic acid or apolypeptide. For example, a nucleic acid sequence is said to “encode” apolypeptide if it can be transcribed and/or translated to produce thepolypeptide or if it can be processed into a form that can betranscribed and/or translated to produce the polypeptide. Such a nucleicacid sequence may include a coding sequence or both a coding sequenceand a non-coding sequence. Thus, the terms “encode,” “encoding” and thelike include an RNA product resulting from transcription of a DNAmolecule, a protein resulting from translation of an RNA molecule, aprotein resulting from transcription of a DNA molecule to form an RNAproduct and the subsequent translation of the RNA product, or a proteinresulting from transcription of a DNA molecule to provide an RNAproduct, processing of the RNA product to provide a processed RNAproduct (e.g., mRNA) and the subsequent translation of the processed RNAproduct.

The term “endogenous” refers to a gene or nucleic acid sequence orsegment that is normally found in a host organism.

The terms “expressible,” “expressed,” and variations thereof refer tothe ability of a cell to transcribe a nucleotide sequence to RNA andoptionally translate the mRNA to synthesize a peptide or polypeptidethat provides a biological or biochemical function.

As used herein, the term “gene” includes a nucleic acid molecule capableof being used to produce mRNA optionally with the addition of elementsto assist in this process. Genes may or may not be capable of being usedto produce a functional protein. Genes can include both coding andnon-coding regions (e.g., introns, regulatory elements, promoters,enhancers, termination sequences and 5′ and 3′ untranslated regions).

The terms “heterologous nucleic acid sequence,” “heterologous nucleotidesequence,” “heterologous polynucleotide,” “foreign polynucleotide,”“exogenous polynucleotide” and the like are used interchangeably torefer to any nucleic acid (e.g., a nucleotide sequence comprising anIRES) which is introduced into the genome of an organism by experimentalmanipulations and may include gene sequences found in that organism solong as the introduced gene contains some modification (e.g., a pointmutation, deletion, substitution or addition of at least one nucleotide,the presence of a endonuclease cleavage site, the presence of a loxPsite, etc.) relative to the viral genomic sequence before themodification.

The terms “heterologous polypeptide,” “foreign polypeptide” and“exogenous polypeptide” are used interchangeably to refer to any peptideor polypeptide which is encoded by an “heterologous nucleic acidsequence,” “heterologous nucleotide sequence,” “heterologouspolynucleotide,” “foreign polynucleotide” and “exogenouspolynucleotide,” as defined above.

The term “mammalian cell” means a cell into which a vector including theattenuated orthopox vector of the invention may be introduced for thepurpose of propagating the poxvirus vector. In one embodiment, the cellis a continuous cell line. It is less imperative that the modified cellis a cell line able to divide continuously. A mammalian or highereukaryotic cell may be modified in accordance with the present inventionand subsequently transformed or immortalised to become a continuouslydividing cell line. However, the cell prior to modification isconveniently a well characterised and continuously dividingbiotechnology compatible continuous cell line known in the art. Suchcells are conveniently available from depositing organisations such asthe American Type Culture Collection (ATCC) or European Collection ofCell Cultures (ECACC).

Suitable mammalian cell lines include, but are not limited to, RK18,BHK, VERO, HBOC-143B, HaCat, HepG2, HeLa, HT1080, HEK-293, RD, COS-7,CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM,myeloma cells (e.g., SB20 cells) and CEMX174 are available, for example,from the ATCC.

Any art recognised genome engineering method for producing modified celllines expressing heterologous genes may be employed. Reference is madeto use of transposon technology to insert genes into the cellular genomeby using piggyBac vectors. However, many method are recognised forintroducing gene into cells, including, without limitation, retrovirustransduction (e.g., MoMLV, etc), lentivirus transduction, plasmidtransfection and integration, other viral system for transducing celllines such as Adenovirus, AAV (Adenovirus Associated Virus), EBV andgenome editing techniques for site specific insertion by homologousrecombination with linear DNA, engineered meganucelases,transcription-activator like effector nucleases (TAL-nuclease),Zinc-finger-nucleases (ZFNs) and CRISPRs.

In one embodiment, the mammalian cell is a CHO cell. Prior art CHO celllines, which do not encode viral host name genes, do not supportmanufacture of vaccinia or vaccinia derivatives substantially unable topropagate in man. As known to those of skill in the art, Chinese hamsterovary (CHO) cells, derived from the ovary of the hamster, Cricetulusgriseus, are the most commonly used mammalian cell for bio-industrialand GMP production of recombinant protein therapeutics, includingantibodies. The popularity of CHO cells for this purpose stems, in part,from their rapid growth and high protein production. As a result, CHOcell lines have been well characterized. Suitable CHO cell lines includewithout limitation A2, A2H, XrS6, CHO-K1, CHO/dhfr, RR-CHO-K1, UT-I,P22, CHO-1C6, Lec1, Lec2, Lec8, Pro-5 an CDKXB1 lines. The CHO-K1 cellline deposited with the ATCC under Accession Number ATCC CLL-61 or ATCCCRL-9618 is frequently employed. The CHO-K1 cell line was derived as asubclone from the parental CHO cell line initiated from a biopsy of anovary of an adult Chinese hamster by Puck T. (1957). The presentspecification describes modified cells other than modified CHO cells.

In some embodiments, the mammalian cell is a human cell, a primate cell,a hamster cell or a rabbit cell.

Cells may be unicellular, or can be grown in tissue culture as liquidcultures, monolayers or the like. Host cells may also be deriveddirectly or indirectly from tissues or may exist within an organismincluding animals.

It will be understood that “inducing” an immune response as contemplatedherein includes eliciting or stimulating an immune response and/orenhancing a previously existing immune response.

As used herein, the term “internal ribosomal entry site” or “IRES”refers to a viral, cellular, or synthetic (e.g., a recombinant)nucleotide sequence which allows for initiation of translation of anmRNA at a site internal to a coding region within the same mRNA or at asite 3′ of the 5′ end of the mRNA, to provide for translation of anoperably linked coding region located downstream of (i.e., 3′ of) theinternal ribosomal entry site. This makes translation independent of the5′ cap structure, and independent of the 5′ end of the mRNA. An IRESsequence provides necessary cis-acting sequences required for initiationof translation of an operably linked coding region.

As used herein the term “isolated” is meant to describe a cell, acompound of interest (e.g., a recombinant poxvirus, a nucleic acidmolecule such as a genome, a polypeptide, etc.) that is in anenvironment different from that in which the compound naturally occurs.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

The term “operably connected” or “operably linked” as used herein refersto a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Forexample, a transcriptional control sequence “operably linked” to acoding sequence refers to positioning and/or orientation of thetranscriptional control sequence relative to the coding sequence topermit expression of the coding sequence under conditions compatiblewith the transcriptional control sequence. In another example, an IRESoperably connected to an orthopox virus coding sequence refers topositioning and/or orientation of the IRES relative to the orthopoxvirus coding sequence to permit cap-independent translation of theorthopox virus coding sequence.

As used here the terms “open reading frame” and “ORF” are usedinterchangeably herein to refer to the amino acid sequence encodedbetween translation initiation and termination codons of a codingsequence. The terms “initiation codon” (e.g., ATG) and “terminationcodon” (e.g., TGA, TAA, TAG) refer to a unit of three adjacentnucleotides (‘codon’) in a coding sequence that specifies initiation andchain termination, respectively, of protein synthesis (mRNAtranslation).

As used herein, the term “parent virus” will be understood to be areference to a virus that is modified to incorporate heterologousnucleic acid sequence to form a recombinant virus of the presentinvention.

The terms “polynucleotide,” “polynucleotide sequence,” “nucleotidesequence,” “nucleic acid” or “nucleic acid sequence as used hereindesignate mRNA, RNA, cRNA, cDNA or DNA. The term typically refers topolymeric form of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of RNA orDNA.

“Polypeptide,” “peptide,” “protein” and “proteinaceous molecule” areused interchangeably herein to refer to molecules comprising orconsisting of a polymer of amino acid residues and to variants andsynthetic analogues of the same. Thus, these terms apply to amino acidpolymers in which one or more amino acid residues are syntheticnon-naturally occurring amino acids, such as a chemical analogue of acorresponding naturally occurring amino acid, as well as tonaturally-occurring amino acid polymers.

As used herein the term “recombinant” as applied to “nucleic acidmolecules,” “polynucleotides” and the like is understood to meanartificial nucleic acid structures (i.e., non-replicating cDNA or RNA;or replicons, self-replicating cDNA or RNA) which can be transcribedand/or translated in host cells or cell-free systems described herein.Recombinant nucleic acid molecules or polynucleotides may be insertedinto a vector. Non-viral vectors such as plasmid expression vectors orviral vectors may be used. The kind of vectors and the technique ofinsertion of the nucleic acid construct according to this invention isknown to the artisan. A nucleic acid molecule or polynucleotideaccording to the invention does not occur in nature in the arrangementdescribed by the present invention. In other words, an heterologousnucleotide sequence is not naturally combined with elements of a parentvirus genome (e.g., promoter, ORF, polyadenylation signal, ribozyme).

As used herein, the term “recombinant virus” will be understood to be areference to a “parent virus” comprising at least one heterologousnucleic acid sequence.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gin, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware.

The terms “signal sequence” or “signal peptide refers to a short (about3 to about 60 amino acids long) peptide that directs co- orpost-translational transport of a protein from the cytosol to certainorganelles such as the nucleus, mitochondrial matrix, and endoplasmicreticulum, for example. For proteins having an ER targeting signalpeptide, the signal peptides are typically cleaved from the precursorform by signal peptidase after the proteins are transported to the ER,and the resulting proteins move along the secretory pathway to theirintracellular (e.g., the Golgi apparatus, cell membrane or cell wall) orextracellular locations. “ER targeting signal peptides,” as used hereininclude amino-terminal hydrophobic sequences which are usuallyenzymatically removed following the insertion of part or all of theprotein through the ER membrane into the lumen of the ER. Thus, it isknown in the art that a signal precursor form of a sequence can bepresent as part of a precursor form of a protein, but will generally beabsent from the mature form of the protein.

“Similarity” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions as defined in Table Abelow. Similarity may be determined using sequence comparison programssuch as GAP (Deveraux et al. 1984, Nucleic Acids Research 12: 387-395).In this way, sequences of a similar or substantially different length tothose cited herein might be compared by insertion of gaps into thealignment, such gaps being determined, for example, by the comparisonalgorithm used by GAP.

TABLE A Exemplary Conservative Amino Acid Substitutions Original ResidueExemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser GlnAsn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln,Glu Met Leu, Ile, Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, PheVal Ile, Leu

Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.

The terms “subject” “patient,” “host” or “individual” usedinterchangeably herein, refer to any subject, particularly a vertebratesubject, and even more particularly a mammalian subject, for whomtherapy or prophylaxis is desired. Suitable vertebrate animals that fallwithin the scope of the invention include, but are not restricted to,any member of the subphylum Chordata including primates (e.g., humans,monkeys and apes, and includes species of monkeys such from the genusMacaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/orrhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well asmarmosets (species from the genus Callithrix), squirrel monkeys (speciesfrom the genus Saimiri) and tamarins (species from the genus Saguinus),as well as species of apes such as chimpanzees (Pan troglodytes)),rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits,hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g.,goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g.,dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks,geese, companion birds such as canaries, budgerigars etc.), marinemammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizardsetc.), and fish. A preferred subject is a human in need of treatment orprophylaxis of a condition. However, it will be understood that theaforementioned terms do not imply that symptoms are present.

The term “transgene” is used herein to describe genetic material thathas been or is about to be artificially introduced into a genome of ahost organism and that is transmitted to the progeny of that host. Insome embodiments, it confers a desired property to a mammalian cell oran orthopox vector into which it is introduced, or otherwise leads to adesired therapeutic or diagnostic outcome.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “wild-type,” “natural,” “native” and the like with respect toan organism, polypeptide, or nucleic acid sequence, that the organismpolypeptide, or nucleic acid sequence is naturally occurring oravailable in at least one naturally occurring organism which is notchanged, mutated, or otherwise manipulated by man.

Variants include nucleic acid molecules sufficiently similar to areferenced molecule or their complementary forms over all or partthereof such that selective hybridisation may be achieved underconditions of medium or high stringency, or which have about 60% to 90%or 90 to 98% sequence identity to the nucleotide sequences defining areferenced poxvirus host range factor over a comparison windowcomprising at least about 15 nucleotides. Preferably the hybridisationregion is about 12 to about 18 nucleobases or greater in length.Preferably, the percent identity between a particular nucleotidesequence and the reference sequence is at least about 80%, or 85%, ormore preferably about 90% similar or greater, such as about 95%, 96%,97%, 98%, 99% or greater. Percent identities between 80% and 100% areencompassed. The length of the nucleotide sequence is dependent upon itsproposed function. Homologs are encompassed. The term “homolog”“homologous genes” or “homologs” refers broadly to functionally andstructurally related molecules including those from other species.Homologs and orthologs are examples of variants.

Nucleic acid sequence identity can be determined in the followingmanner. The subject nucleic acid sequence is used to search a nucleicacid sequence database, such as the GenBank database (accessible at website www.nchi.nln.nih.gov/blast/), using the program BLASTM version 2.1(based on Altschul et al. (1997) Nucleic Acids Research 25:3389-3402).The program is used in the ungapped mode. Default filtering is used toremove sequence homologies due to regions of low complexity. The defaultparameters of BLASTM are used.

Amino acid sequence identity can be determined in the following manner.The subject polypeptide sequence is used to search a polypeptidesequence database, such as the GenBank database (accessible at web sitehttp://www.ncbi.nln.nih.gov/blast/), using the BLASTP program. Theprogram is used in the ungapped mode. Default filtering is used toremove sequence homologies due to regions of low complexity. The defaultparameters of BLASTP are utilized. Filtering for sequences of lowcomplexity may use the SEG program.

Preferred sequences will hybridise under stringent conditions to areference sequence or its complement. The term “hybridize understringent conditions”, and grammatical equivalents thereof, refers tothe ability of a nucleic acid molecule to hybridize to a target nucleicacid molecule (such as a target nucleic acid molecule immobilized on aDNA or RNA blot, such as a Southern blot or Northern blot) under definedconditions of temperature and salt concentration. With respect tonucleic acid molecules greater than about 100 bases in length, typicalstringent hybridization conditions are no more than 25° C. to 30° C.(for example, 10° C.) below the melting temperature (Tm) of the nativeduplex (see generally, Sambrook et al., (supra); Ausubel et al.,(1999)). Tm for nucleic acid molecules greater than about 100 bases canbe calculated by the formula Tm=81.5+0.41% (G+C−log (Na⁺)). With respectto nucleic acid molecules having a length less than 100 bases, exemplarystringent hybridization conditions are 5° C. to 10° C. below Tm.

The term “deletion” in the present context removal of all or part of thecoding region of the target gene. The term also encompasses any form ofmutation or transformation which ablates gene expression of the targetgene or ablates or substantially down regulates the level or activity ofthe encoded protein.

Reference to “gene” includes DNA corresponding to the exons or the openreading frame of a gene. Reference herein to a “gene” is also taken toinclude: a classical genomic gene consisting of transcriptional and/ortranslational regulatory sequences and/or a coding region and/ornon-translated sequences (i.e. introns, 5′- and 3′-untranslatedsequences); or mRNA or cDNA corresponding to the coding regions (i.e.exons) and 5′- and 3′-untranslated sequences of the gene.

By “regulatory element” or “regulatory sequence” is meant nucleic acidsequences (e.g., DNA) necessary for expression of an operably linkedcoding sequence in a particular host cell. The regulatory sequences thatare suitable for prokaryotic cells for example, include a promoter, andoptionally a cis-acting sequence such as an operator sequence and aribosome binding site. Control sequences that are suitable foreukaryotic cells include promoters, polyadenylation signals,transcriptional enhancers, translational enhancers, leader or trailingsequences that modulate mRNA stability, as well as targeting sequencesthat target a product encoded by a transcribed polynucleotide to anintracellular compartment within a cell or to the extracellularenvironment.

Chimeric constructs suitable for effecting the present modifiedmammalian cells comprise a nucleic acid sequence encoding an orthopoxhost range factor, which is operably linked to a regulatory sequence.The regulatory sequence suitably comprises transcriptional and/ortranslational control sequences, which will be compatible for expressionin the cell. Typically, the transcriptional and translational regulatorycontrol sequences include, but are not limited to, a promoter sequence,a 5′ non-coding region, a cis-regulatory region such as a functionalbinding site for transcriptional regulatory protein or translationalregulatory protein, an upstream open reading frame, ribosomal-bindingsequences, transcriptional start site, translational start site, and/ornucleotide sequence which encodes a leader sequence, termination codon,translational stop site and a 3′ non-translated region. Constitutive orinducible promoters as known in the art are contemplated. The promotersmay be either naturally occurring promoters, or hybrid promoters thatcombine elements of more than one promoter.

Promoter sequences contemplated may be native to mammalian cells or maybe derived from an alternative source, where the region is functional inthe chosen organism. The choice of promoter will differ depending on theintended host cell. For example, promoters which could be used forexpression in mammalian cells include the metallothionein promoter,which can be induced in response to heavy metals such as cadmium, theβ-actin promoter as well as viral promoters such as the SV40 large Tantigen promoter, human cytomegalovirus (CMV) immediate early (IE)promoter, Rous sarcoma virus LTR promoter, the mouse mammary tumor virusLTR promoter, the adenovirus major late promoter (Ad MLP), the herpessimplex virus promoter, and a HPV promoter, particularly the HPVupstream regulatory region (URR), among others. All these promoters arewell described and readily available in the art.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described for example in Dijkema et al. (1985) EMBO J.4:761, the enhancer/promoter derived from the long terminal repeat (LTR)of the Rous Sarcoma Virus, as described for example in Gorman et al.,(1982) Proc. Natl. Acad. Sci. USA 79:6777 and elements derived fromhuman CMV, as described for example in Boshart et al. (1985) Cell41:521, such as elements included in the CMV intron A sequence.

The chimeric construct may also comprise a 3′ non-translated sequence. A3′ non-translated sequence refers to that portion of a gene comprising aDNA segment that contains a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing or geneexpression. The polyadenylation signal is characterized by effecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. Polyadenylation signals are commonly recognized by thepresence of homology to the canonical form 5′ AATAAA-3′ althoughvariations are not uncommon. The 3′ non-translated regulatory DNAsequence preferably includes from about 50 to 1,000 nts and may containtranscriptional and translational termination sequences in addition to apolyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression.

In some embodiments, the chimeric construct further contains aselectable marker gene to permit selection of cells containing theconstruct. Selection genes are well known in the art and will becompatible for expression in the cell of interest.

In one embodiment, expression of the orthopox structural or assemblygene is under the control of a promoter. In one non-limiting embodimentthe promoter is a cellular constitutive promoter, such as human EF1alpha (human elongation factor 1 alpha gene promoter), DHFR(dihydrofolate reductase gene promoter) or PGK (phosphoglycerate kinasegene promoter) that direct expression of a sufficient level of CP77 tosustain viral propagation in the absence of significant toxic effects onthe host cell. Promoters may also be inducible, such as the cellularinducible promoter, MTH (from a metallothionein gene) viral promotersare also employed in mammalian cells, such as CMV, RSV, SV-40, and MoU3.

In a first aspect the present invention provides a modified mammaliancell in which the genome of the cell is modified to comprise a sequenceencoding CP77 under the control of a promoter such that the modifiedcell line sustains propagation of a poxvirus that is less able or unableto propagate in the unmodified cell.

In a second aspect the present invention provides a process forpropagating an orthopoxvirus that does not propagate in CHO cells, theprocess comprising propagating the poxvirus in vitro in a mammalian cellline wherein the cell line is modified to encode and express CP77 underthe control of a promoter.

In an embodiment the genome of the cell further comprises a sequenceencoding D13L under the control of a promoter and/or further comprises asequence encoding K1L under the control of a promoter.

In another embodiment the cell is a continuous cell line, preferably aCHO cell.

In other embodiments the cell may be a human cell, a primate cell, ahamster cell or a rabbit cell.

In another embodiment the CP77 gene, D13L gene and/or the K1L gene isunder the control of a mammalian promoter.

In another embodiment the expression of the CP77 gene supportspropagation of the virus to generate virus yields equivalent to thatobserved in a permissive cell line and preferably supports a virusreplication amplification ratio of more than 500.

In another embodiment the CP77, D13L and/or K1L is/are encoded by acontiguous sequence of nucleotides codon optimised for expression inmammalian cells.

An example K1L sequence is provided in SEQ ID NO:6 and SEQ ID NO:7.

The specification relates broadly to modified (recombinant ortransformed) cells and to a process for in vitro manufacture of virusvectors in cultured higher eukaryotic host cells, wherein the cell isgenetically modified in such a way as to enhance or facilitatemultiplication of the virus vector within or via a population of thecells in vitro. In one non-limiting embodiment, the specificationprovides for the propagation of vaccinia-based poxviruses in modifiedChinese hamster ovary (CHO) cells.

As known to those of skill in the art, Chinese hamster ovary (CHO)cells, derived from the ovary of the hamster, Cricetulus griseus, arethe most commonly used mammalian cell for bio-industrial and GMPproduction of recombinant protein therapeutics, including antibodies.The popularity of CHO cells for this purpose stems, in part, from theirrapid growth and high protein production. As a result, CHO cell lineshave been well characterized. Suitable CHO cell lines include withoutlimitation A2, A2H, XrS6, CHO-K1, CHO/dhfr, RR-CHO-K1, UT-I, P22,CHO-1C6, Lec1, Lec2, Lec8, Pro-5 an CDKXB1 lines. The CHO-K1 cell linedeposited with the ATCC under Accession Number ATCC CLL-61 or ATCCCRL-9618 is frequently employed. The CHO-K1 cell line was derived as asubclone from the parental CHO cell line initiated from a biopsy of anovary of an adult Chinese hamster by Puck T. (1957). The presentspecification describes modified cells other than modified CHO cells.

To maximise the yield of virus produced, cell lines such as CHO cellsmay be adapted to suspension culture using standard techniques.Reference to a recombinant or modified cell includes its progeny. Cellsmay be sold in any form, including frozen or in liquid suspension form.Cells may be sold infected with a poxvirus vector.

Reference herein to CP77 means the cowpox host range gene referred to inSphener et al. (1988), and Hsiao et al. (2006) and to functionalorthologs and modified forms thereof able as determined herein tosupport pox viral growth when expressed as a heterologous gene in thegenome of a mammalian host cell. Reference to “modified forms” includesa variation (such as a deletion, substitution or addition) from thewild-type or reference sequence. A reference nucleotide sequence isprovided in SEQ ID NO: 1. Modified forms share at least 80% sequenceidentity over the coding region or one or more portions thereofcomprising at least 200 contiguous basepairs. Modified forms includecodon optimised forms as described herein that are optimised forexpression in a mammalian or other higher eukaryotic cells including CHOcells. Reference to CP77 includes orthologs i.e., genes with the samefunction present in other species or identified by different names. Thecowpox host range factor, CP77 is also referred to as VHR1, CHOhr, andCPXV025. The CHOhr/CP77 gene in the Western Reserve (WR) strain ofvaccinia is substantially fragmented and fails to produce a functionalfactor. CP77 is absent in MVA and the Copenhagen strain.

Reference herein to “CP77”, “D13L” and “K1L” includes functionalorthologs and functional variants. “Functional” refers to the hereindescribed quality of expression, (i.e., transcription and translation)directed from the mammalian genome of a cultured cell, to supportpoxvirus propagation within the expressing cell cytoplasm. The term“propagate” includes intracellular propagation and intercellularpropagation and encompasses the production of mature viral particles.

In one embodiment, a population of the modified cells sustainspropagation of a poxvirus that is less able or substantially unable topropagate in an unmodified control cell. The inability to propagatemeans that cell to cell or subject to subject transmission does nottypically occur.

Reference to “unmodified control cell” includes the modified cell as itexisted prior to modification to encode at least one viral host rangefactor selected from the group consisting of CP77, K1L and SPI-1 and toexpress same from its genome under control of a promoter, as well asother suitable control cells known in the art.

For example, poxviruses such as MVA and vaccinia are unable to propagatein CHO cells. However, as surprisingly determined herein, CHO cells thatare recombinantly modified to express CP77 from their genome under thecontrol of a promoter are able to support propagation of not onlyvaccinia but also derivatives of vaccinia such as MVA. After propagationin the subject modified CHO-CP77 cells, MVA and vaccinia are stillunable to propagate in unmodified CHO cells or other suitable control.

Reference herein to “poxvirus” includes a recombinant poxvirus vectorencoding a heterologous molecule of interest (such as an antigen ofinterest) as a medicament, prophylactic, diagnostic or therapeuticagent. Such recombinant poxvirus vectors are typically for use asvaccines against non-poxviral induced diseases or conditions. Also, theterm poxvirus includes isolated poxviruses and derivatives thereofproposed for use as a therapeutic or prophylactic vaccine against apoxvirus infection, such as variola or small pox infection.

Reference herein to “vaccinia-based” includes vaccinia and derivativesand modified forms of vaccinia virus. Vaccinia based derivativesinclude, but are in no way limited to, MVA and NYVAC. Reference to MVAand NYVAC includes strains or derivates of these poxviruses known in theart. Modified forms may have modifications in one to ten or more genes.The term “modification” is intended to mean a variation (such as adeletion, substitution or addition) from the wild-type or referencesequence. Reference to “attenuated” includes poxvirus that do notpropagate or which propagate to a substantially lesser degree in arelevant subject compared to a non-attenuated form of the same virus.The term also includes viruses that are non-pathogenic in a subject.

In one embodiment, the cell is a continuous cell line. It is lessimperative that the modified cell is a cell line able to dividecontinuously. A mammalian or higher eukaryotic cell may be modified inaccordance with the present invention and subsequently transformed orimmortalised to become a continuously dividing cell line. However, thecell prior to modification is conveniently a well characterised andcontinuously dividing biotechnology compatible continuous cell lineknown in the art. Such cells are conveniently available from depositingorganisations such as the American Type Culture Collection (ATCC) orEuropean Collection of Cell Cultures (ECACC).

Suitable mammalian cell lines include, but are not limited to, RK18,BHK, VERO, HBOC-143B, HaCat, HepG2, HeLa, HT1080, HEK-293, RD, COS-7,CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM,myeloma cells (e.g., SB20 cells) and CEMX174 are available, for example,from the ATCC.

In one embodiment, the cell is a CHO cell. Prior art CHO cell lines,which do not encode viral host name genes, do not support manufacture ofvaccinia or vaccinia derivatives substantially unable to replicate inman.

In some embodiments, the cell is a human cell, a primate cell, a hamstercell or a rabbit cell.

Chimeric constructs suitable for effecting the present modifiedmammalian cells comprise a nucleic acid sequence encoding a poxviralhost range factor, which is operably linked to a regulatory sequence.The regulatory sequence suitably comprises transcriptional and/ortranslational control sequences, which will be compatible for expressionin the cell. Typically, the transcriptional and translational regulatorycontrol sequences include, but are not limited to, a promoter sequence,a 5′ non-coding region, a cis-regulatory region such as a functionalbinding site for transcriptional regulatory protein or translationalregulatory protein, an upstream open reading frame, ribosomal-bindingsequences, transcriptional start site, translational start site, and/ornucleotide sequence which encodes a leader sequence, termination codon,translational stop site and a 3′ non-translated region. Constitutive orinducible promoters as known in the art are contemplated. The promotersmay be either naturally occurring promoters, or hybrid promoters thatcombine elements of more than one promoter.

Promoter sequences contemplated may be native to mammalian cells or maybe derived from an alternative source, where the region is functional inthe chosen organism. The choice of promoter will differ depending on theintended host cell. For example, promoters which could be used forexpression in mammalian cells include the metallothionein promoter,which can be induced in response to heavy metals such as cadmium, theβ-actin promoter as well as viral promoters such as the SV40 large Tantigen promoter, human cytomegalovirus (CMV) immediate early (IE)promoter, Rous sarcoma virus LTR promoter, the mouse mammary tumor virusLTR promoter, the adenovirus major late promoter (Ad MLP), the herpessimplex virus promoter, and a HPV promoter, particularly the HPVupstream regulatory region (URR), among others. All these promoters arewell described and readily available in the art.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described for example in Dijkema et al. (1985) EMBO J.4:761, the enhancer/promoter derived from the long terminal repeat (LTR)of the Rous Sarcoma Virus, as described for example in Gorman et al.,(1982) Proc. Natl. Acad. Sci. USA 79:6777 and elements derived fromhuman CMV, as described for example in Boshart et al. (1985) Cell41:521, such as elements included in the CMV intron A sequence.

The chimeric construct may also comprise a 3′ non-translated sequence. A3′ non-translated sequence refers to that portion of a gene comprising aDNA segment that contains a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing or geneexpression. The polyadenylation signal is characterized by effecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. Polyadenylation signals are commonly recognized by thepresence of homology to the canonical form 5′ AATAAA-3′ althoughvariations are not uncommon. The 3′ non-translated regulatory DNAsequence preferably includes from about 50 to 1,000 nts and may containtranscriptional and translational termination sequences in addition to apolyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression.

In some embodiments, the chimeric construct further contains aselectable marker gene to permit selection of cells containing theconstruct. Selection genes are well known in the art and will becompatible for expression in the cell of interest.

In one embodiment, expression of the viral host range gene is under thecontrol of a promoter. In one non-limiting embodiment the promoter is acellular constitutive promoter, such as human EF1 alpha (humanelongation factor 1 alpha gene promoter), DHFR (dihydofolate reductasegene promoter) or PGK (phosphoglycerate kinase gene promoter) thatdirect expression of a sufficient level of CP77 to sustain viralpropagation in the absence of significant toxic effects on the hostcell. Promoters may also be inducible, such as the cellular induciblepromoter, MTH (from a metallothionein gene) viral promoters are alsoemployed in mammalian cells, such as CMV, RSV, SV4, and MoU3.

Conveniently, in one embodiment, the expression of the viral host rangegene supports propagation of the virus to generate virus yieldsequivalent to that observed in permissive cell lines. The expression ofthe viral host range gene, for example, supports a virus replicationamplification ratio of more than 500. The expression of the viral hostrange gene supports viral propagation in the absence of significant hostcell toxicity. Significant host cell toxicity refers to a level of viralhost range factor expression that reduces viral yield due to prematurehost cell death or failure to divide. The skilled artisan is familiarwith methods for qualitatively or quantitatively assessing host cellparameters such as host cell survival and multiplication, and viralparameters, such as viral host range gene expression, viral replicationand viral yield.

In one embodiment, the poxvirus is a chordopox virus, other than anorthopox virus that encodes a functional CP77 or CP77 ortholog. Cowpoxvirus encodes CP77 and is therefore not encompassed in this aspect.Orthopox viruses include, buffalopox virus, cowpox virus, camelpoxvirus, ectromelia virus, monkeypox virus, rabbitpox virus, racconpoxvirus, teterapox virus, vaccinia virus, volepox virus, skunkpox virus,and Uasin Gishu disease virus of horses. Other genus include theparapoxviruses, avipoxviruses, capripoxviruses, leporipoxviruses,swinepoxviruses, molluscipoxviruses and yatapoxviruses.

In one embodiment, the poxvirus is MVA or a derivative of MVA that issubstantially unable to replicate in man/a subject.

In one embodiment, the poxvirus is vaccinia or an derivative of vacciniathat is substantially non-replicative in vivo in man.

In another embodiment, the poxvirus is suitable for use as a poxviralvaccine.

In one further embodiment, the poxvirus is a recombinant poxviral vectorwhich encodes and expresses a heterologous molecule of interest, such asan antigen of medical interest, wherein the recombinant poxviral vectoris for use as a diagnostic, therapeutic or prophylactic agent in asubject.

Reference herein to K1L means the gene described by Shisler and Jin(2004) and orthologs or modified forms thereof.

Reference herein to SPI-1 means the host range gene described by Brookeset al. (1995) or orthologs and modified forms thereof.

In some embodiments, and for the avoidance of doubt, the instantenhanced viral propagative process does not require addition of genes tothe poxviral genome. This does not, of course, exclude modifications ofviral vectors for other purposes, such as, without limitation, to encodeheterologous molecules as antigens of interest for vaccine purposes orto engender an immune response in a subject.

Transcription of the poxviral host range gene from within the host cellnucleus and translation of the encoded product takes place in theinfected cell and is sufficient for pox viral propagation in the hostcell cytoplasm. Without limitation to any particular mode of action, itis proposed that CP77 is a viral protection agent.

In one illustrative embodiment, the poxviral host range gene expressedby the infected cell line is CP77. As shown in Example 4, when a CHOcell nucleus is modified to encode and express CP77 it is able tosustain viral amplification as if it were a permissive cell line such as143B. Typically, confluent plaques are observed within two days frominfection.

In another embodiment, the level of viral propagation in the modifiedcell provides an amplification ratio of at least 10 to 5000.Amplification ratios are, in some embodiments, between 500 and 3000, orbetween 1000 and 4000.

In another embodiment, the promoter driving expression of theheterologous pox viral host range gene provides a level of pox viralhost range heterologous gene expression in the cell. The level of CP77expression can be similar to or exceed that produced by cowpox virus inpermissive cells.

In another embodiment, the promoter driving expression of theheterologous pox viral host range gene provides a level of heterologousgene expression in the cell sufficient to allow viral propagation atleast to the level of viral propagation in a permissive cell.

In one embodiment, poxviral production in a CHO cell line is equal to orexceeds the level of poxviral production in a positive control cell.

In one embodiment, the level of MVA viral production in CHO cells issubstantially equal to or exceeds the level of MVA viral production inCEF cells.

In one embodiment, CP77, K1L and/or SPI-1 is/are encoded by a contiguoussequence of nucleotides that is codon optimised for expression inmammalian cells.

As described further herein, the codon optimised nucleic acid sequenceencoding CP77 may have less than 80% or less than 75% nucleotidesequence identity to the sequence encoding the cowpox CP77 protein ofthe Brighton Red strain (UniprotKB/Swiss-Prot:P12932.1). In someembodiments the codon optimised sequence has the sequence set forth inSEQ ID NO: 1 or is a functional variant comprising a nucleic acidsequence that has at least 70% sequence identity to the sequence setforth in SEQ ID NO: 1. In some embodiments, the CP77 virus host rangefactor has an amino acid sequence set forth in SEQ ID NO: 2 or has atleast 70% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:2.

In some embodiments, a kit is contemplated comprising or consistingessentially of a population, such as a clonal population, of modifiedmammalian cells expressing CP77 from their genome as described herein.In some embodiments, the modified cell does not contain a vacciniavirus.

The present description further describes a process for or method ofmanufacturing a poxvirus that does not propagate in CHO cells, theprocess comprising propagating the poxvirus in vitro in a mammalian cellline wherein the cell line is modified to encode and express CP77 underthe control of a promoter. The process may further comprise isolatingviral particles.

The cell line is conveniently a mammalian cell line known to those ofskill in the art to be suitable for the manufacture of a medicament ortherapeutic, diagnostic or prophylactic agent.

The specification describes a modified CHO cell, wherein the CHO cell ismodified to encode CP77 and express same from its genome under controlof a promoter.

In one embodiment, the modified CHO cell line sustains propagation of avirus that is less able or unable to propagate in an unmodified controlCHO cell which is one that does not express CP77.

In one embodiment, the virus is an orthopox virus other than an orthopoxvirus that encodes CP77. As known in the art, cowpox virus is a poxvirus that encodes CP77.

In some embodiments, the virus is vaccinia or a derivative of vacciniathat is substantially non-replicative in vivo in man/a subject.

In some embodiments, the virus is MVA.

In another embodiment, the specification provides a method ofpropagating a poxvirus which is substantially non-replicative in man,the method comprising: (i) culturing a CHO cell which has beentransformed to express CP77; and (ii) infecting the cultured CHO cellfrom (i) with the poxvirus which is substantially non-replicative inman.

In another embodiment, the specification provides a method ofpropagating a poxvirus which is substantially non-replicative in man,the method comprising: (i) culturing a CHO cell which has beentransformed to express CP77 and D13L and/or K1L, and (ii) infecting thecultured CHO cell from (i) with the poxvirus which is substantiallynon-replicative in man.

In another embodiment, the specification provides a method ofpropagating MVA, the method comprising: (i) culturing a CHO cell whichhas been transformed to express CP77 and D13L and/or K1L and (ii)infecting the cultured CHO cell from (i) with MVA.

In another embodiment, the specification provides a method ofpropagating, a vaccinia derivative which is substantiallynon-replicative in man, the method comprising: (i) culturing a CHO cellwhich has been transformed to express CP77 and D13L and/or K1L and (ii)infecting the cultured CHO cell from (i) with the vaccinia derivative.

In another embodiment, the specification provides a method ofpropagating MVA encoding a heterologous protein, the method comprising:(i) culturing a CHO cell which has been transformed to express CP77 andD13L and/or K1L and (ii) infecting the cultured CHO cell from (i) withthe MVA.

In another embodiment, the specification provides a method ofpropagating, a vaccinia derivative encoding a heterologous protein,which is substantially non-replicative in man, the method comprising:(1) culturing a CHO cell which has been transformed to express CP77 andD13L and/or K1L and (ii) infecting the cultured CHO cell from (i) withthe vaccinia derivative.

In another embodiment, the present specification provides anartificially created vector, polynucleotide or plasmid comprising thenucleic acid sequence of a virus host range gene operably connected toregulatory elements such as a promoter for expression in a mammaliancell line. In some embodiments, the virus gene is cowpox ankyrin repeatdomain-containing protein CP77 gene (UniProtKBSwiss-Prot P12932.1[025LBR CP77 protein]. In one embodiment the virus host range gene, suchas CP77, is codon optimised for expression in a mammalian cell line.Suitable vectors and plasmids are known in the art.

In one embodiment, the polynucleotide encodes CP77. In some embodiments,the polynucleotide comprises the nucleotide sequence set forth in SEQ IDNO: 1 (codon optimised for expression in mammalian cells such as CHO).In some embodiments, the isolated polynucleotide comprises thenucleotide sequence set forth in SEQ ID NO:1 or a variant thereof thatencodes the amino acid sequence set out in SEQ ID NO: 2.

In another embodiment, the present specification described atransposition delivery vector for stable insertion of a virus host rangegene into a mammalian cell.

The present description also provides a method of transforming amammalian or higher eukaryotic culture cell which is substantiallynon-permissive to a virus, into a cell which is permissive to the virus,the method comprising transforming the cell to express CP77.

In some embodiments, the method includes transfecting the cell with avector capable of directing expression of an encoded CP77 under thecontrol of a mammalian promoter.

In some embodiments the vector is a transposition delivery vectorencoding CP77 under the control of a mammalian promoter.

In one non-limiting embodiment the promoter is a cellular constitutivepromoter, such as human EF1 alpha (human elongation factor 1 alpha genepromoter), DHFR (dihydofolate reductase gene promoter) or PGK(phosphoglycerate kinase gene promoter) that direct expression of asufficient level of CP77 to sustain viral propagation in the absence ofsignificant toxic effects on the host cell. Promoters may also beinducible, such as the cellular inducible promoter, MTH (from ametallothionein gene) viral promoters are also employed in mammaliancells, such as CMV, RSV, SV4, and MoU3.

In some embodiments, the cell is a CHO cell.

In some embodiments the virus is a poxvirus.

In some embodiments the poxvirus is a vaccinia derivative which isnon-pathogenic or non-replicating in man.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample an “isolated polynucleotide” or an “isolated polypeptide” andthe like, as used herein, refer to in vitro isolation and/orpurification of a polynucleotide or polypeptide molecule from itsnatural cellular environment, and from association with other componentsof the cell. Without limitation, an isolated composition, complex,polynucleotide, peptide, or polypeptide can refer to a native sequencethat is isolated by purification or to a sequence that is produced byrecombinant or synthetic means.

Variants include nucleic acid molecules sufficiently similar to areferenced molecule or their complementary forms over all or partthereof such that selective hybridisation may be achieved underconditions of medium or high stringency, or which have about 60% to 90%or 90 to 98% sequence identity to the nucleotide sequences defining areferenced poxvirus host range factor over a comparison windowcomprising at least about 15 nucleotides. Preferably the hybridisationregion is about 12 to about 18 nucleobases or greater in length.Preferably, the percent identity between a particular nucleotidesequence and the reference sequence is at least about 80%, or 85%, ormore preferably about 90% similar or greater, such as about 95%, 96%,97%, 98%, 99% or greater. Percent identities between 80% and 100% areencompassed. The length of the nucleotide sequence is dependent upon itsproposed function. Homologs are encompassed. The term “homolog”“homologous genes” or “homologs” refers broadly to functionally andstructurally related molecules including those from other species.Homologs and orthologs are examples of variants.

Nucleic acid sequence identity can be determined in the followingmanner. The subject nucleic acid sequence is used to search a nucleicacid sequence database, such as the GenBank database (accessible at website http://www.ncbi.nln.nih.gov/blast/), using the program BLASTMversion 2.1 (based on Altschul et al. (1997) Nucleic Acids Research25:3389-3402). The program is used in the ungapped mode. Defaultfiltering is used to remove sequence homologies due to regions of lowcomplexity. The default parameters of BLASTM are used.

Amino acid sequence identity can be determined in the following manner.The subject polypeptide sequence is used to search a polypeptidesequence database, such as the GenBank database (accessible at web sitehttp://www.ncbi.nln.nih.gov/blast/), using the BLASTP program. Theprogram is used in the ungapped mode. Default filtering is used toremove sequence homologies due to regions of low complexity. The defaultparameters of BLASTP are utilized. Filtering for sequences of lowcomplexity may use the SEG program.

The term “hybridize under stringent conditions”, and grammaticalequivalents thereof, refers to the ability of a nucleic acid molecule tohybridize to a target nucleic acid molecule (such as a target nucleicacid molecule immobilized on a DNA or RNA blot, such as a Southern blotor Northern blot) under defined conditions of temperature and saltconcentration. With respect to nucleic acid molecules greater than about100 bases in length, typical stringent hybridization conditions are nomore than 25° C. to 30° C. (for example, 10° C.) below the meltingtemperature (Tm) of the native duplex (see generally, Sambrook et al.,(supra); Ausubel et al., (1999)). Tm for nucleic acid molecules greaterthan about 100 bases can be calculated by the formula Tm=81.5+0.41%(G+C−log(Na⁺)). With respect to nucleic acid molecules having a lengthless than 100 bases, exemplary stringent hybridization conditions are 5°C. to 10° C. below Tm.

By “vector” is meant a polynucleotide molecule, suitably a DNA moleculederived, for example, from a plasmid, bacteriophage, yeast, virus,mammal, avian, reptile or fish into which a polynucleotide can beinserted or cloned. A vector preferably contains one or more uniquerestriction sites and can be capable of autonomous replication in adefined host cell including a target cell or tissue or a progenitor cellor tissue thereof, or be integrable with the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectorcan be an autonomously replicating vector, i.e., a vector that exists asan extrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector can contain any means for assuring self-replication.Alternatively, the vector can be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. A vector system cancomprise a single vector or plasmid, two or more vectors or plasmids,which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon. The choice of the vector will typicallydepend on the compatibility of the vector with the host cell into whichthe vector is to be introduced. The vector can also include a selectionmarker such as an antibiotic resistance gene that can be used forselection of suitable transformants. Examples of such resistance genesare known to those of skill in the art.

Reference to “gene” includes cDNA corresponding to the exons of a gene.Reference herein to a “gene” is also taken to include: a classicalgenomic gene consisting of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e. introns, 5′- and 3′-untranslated sequences); or mRNA orcDNA corresponding to the coding regions (i.e. exons) and 5′- and3′-untranslated sequences of the gene.

By “regulatory element” or “regulatory sequence” is meant nucleic acidsequences (e.g., DNA) necessary for expression of an operably linkedcoding sequence in a particular host cell. The regulatory sequences thatare suitable for prokaryotic cells for example, include a promoter, andoptionally a cis-acting sequence such as an operator sequence and aribosome binding site. Control sequences that are suitable foreukaryotic cells include promoters, polyadenylation signals,transcriptional enhancers, translational enhancers, leader or trailingsequences that modulate mRNA stability, as well as targeting sequencesthat target a product encoded by a transcribed polynucleotide to anintracellular compartment within a cell or to the extracellularenvironment.

Complementary sequences and parts of these sequences are encompassed.The term “complement” and “complementary” when used in connection with anucleic acid molecule refers to the complementary nucleic acid sequenceas determined by Watson-Crick base pairing. For example, the complementof the nucleic acid sequence 5′CCATG3′ is 5′CATGG3′.

The phrase “hybridizing specifically to” and the like refer to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The terms “subject” or “individual” or “patient”, used interchangeablyherein, refer to any subject, particularly a vertebrate subject, andeven more particularly a mammalian subject, for whom therapy orprophylaxis is desired. Suitable vertebrate animals that fall within thescope of the invention include, but are not restricted to, primates,avians, livestock animals (e.g., sheep, cows, horses, donkeys, pigs),laboratory test animals (e.g., rabbits, mice, rats, guinea pigs,hamsters), companion animals (e.g., cats, dogs) and captive wild animals(e.g., foxes, deer, dingoes). A preferred subject is a primate such as ahuman in need of treatment or prophylaxis. However, it will beunderstood that the aforementioned terms do not imply that symptoms arepresent.

The various embodiments enabled herein are further described by thefollowing non-limiting examples.

Example 1 VACV-COP Fails to Grow in CHO Cells

Materials and Reagents

VACV-COP, VSS02, SEM120213, Titre: 1.6×10(8) pfu/mL

Vero: WHO-VERO-MCB passage No 141, Aug. 8, 2005, Virax Holdings Limited

CHO: SA-Pathology Jul. 5, 2004

Growth medium: RPMI, 10% FBS, Pen/Strep

Maintenance medium: RPMI, 2% FBS, Pen/Strep

CHO and Vero cells were cultured to confluency in one 6-Well plate(6-WP) per cell line. Each well was infected with 4×10(4) pfu ofVACV-COP VSSO2 for 45 minute at room temperature and then incubated at37° C./5% CO2 thereafter. From each cell line, the contents of 2 wellswhere harvested and pooled at 24 h, 48 h, 72 h post infection. Viralextracts were made by freeze thawing three times and storing at −80° C.until ready for titrations. Each extract was titred using Vero cells asdescribed in the Protocol described in Example 4.

After freeze-thawing a homogenation probe may be used to break up theselarge insoluble clumps. Each well to be harvested contains 2 mL of MM.For each time point, 2 wells were pooled to give a total volume of 4 mLper time point. TE buffer may be added to give a total volume of 6 mLper time point.

The titration results, viral yield results and production yields aretabulated in Table 1. The results show that VACV-COP is unable topropagate from a low moi in CHO cells, unlike Vero cells where viralproduction increases with time. VACP-COP is non-permissive in CHO cells.

Example 2 Multiple-Step Growth for VACV+PH22 [CP77] in CHO VersesVero-CP77 is Active in VACV

The propagation potential in CHO of a recombinant VACV-COP expressingthe cow pox virus BRO25L gene encoding CP77 (VACV-PH22 [CP77]) wasdetermined in comparison to Vero cells in a multistep growth study.

VACV-PH22 is a recombinant vaccinia virus Copenhagen strain expressingthe native 025L ORF from the Brighton strain of cowpox virus that codesfor the CHO host range protein, CP77. This ORF in vaccinia is also underthe control of the BRO25L native promoter. The native BRO25L gene(native promoter and ORF) was cloned to create pPH22 which also codesfor a red fluorescent protein, DsRed-Express2. pPH22 is an integrationvector that will insert the BRO25L gene and the DsRed gene into the B19RORF of VACV-COP, which codes for the soluble and cell surface IFNalpha/beta receptor protein. Insertion of the BRO25L and DsRed intoVACV-COP was achieved by homologous recombination as a result ofinfecting CHO with a low multiplicity of VACV-COP and transfection withpPH22. Only virus containing the BRO25L gene will be amplified furtherin CHO cells which can be visually verified by the presence of redfluorescent infected cells or plaques.

Virus extracted 3 days after homologous recombination was amplifiedthree times in CHO and then titred in Vero cells before using in thismultistep growth study in CHO cells.

Materials and Reagents

VACV-COP, VSS02, SEM120213, Titre: 1.6×108 pfu/mL

VACV-PH22 [CP77], Titre: 8×106 pfu/mL

Vero: WHO-VERO-MCB passage No 141, Aug. 8, 2005

CHO: SA-Pathology Jul. 5, 2004

Growth medium: RPMI, 10% FBS, Pen/Strep for CHO and Vero

Maintenance medium: RPMI, 2% FBS, Pen/Strep for CHO and Vero

CHO and Vero cells were cultured to confluency in 2× T25 flasks per cellline. 1 flask of each cell line was infected with 1×10⁵ pfu of VACV-COPVSS02 and one each with 1×10⁵ pfu of VACV-PH22 for 45 minutes at roomtemperature and then incubated at 37° C./5% CO2 thereafter. From eachcell line, the contents of each flask where harvested 96 h postinfection. Viral extracts were made by freeze thawing three times andstoring at −80° C. until ready for titrations. Each extract was titredusing Vero cells as described in the protocol detailed in Example 4 in24 well plate formats.

Infections were done in T25 flask, 2 flask per cell line where flask 1was infection with VACV-COP and flask 2 infection with VACV-PH22.Harvesting was only performed at 96 h post infection. The results aretabulated in Table 2. From Table 2 it can be seen that: CHO cells cannotsupport VACV-COP viral progeny production. This confirms the resultsdescribed in Example 1. Further, VACV-COP was viable as infection of apermissive cell line, Vero cells in this study, amplified the inoculumlevel to over 200 fold, therefore the results seen in CHO where due tohost cell restriction. However, when the CP77 was expressed by arecombinant vaccinia virus, VACV-PH22, amplification of the inoculum wasachieved by about 700 fold. Expression of the CP77 did not limitvaccinia host range to only CHO cells, as infection of vero cells withVACV-PH22 also amplified the inoculum. However, the level ofamplification may not be as good as vaccinia without CP77. Since thestandard error of the titration results are so large the difference inyield may not be significant.

These results show that CP77 expressed from the viral genome is morethan adequate at enabling VACV-COP to amplify in the non-permissive CHOcell line by producing yields similar to that expected from a permissivecell substrate such as Vero.

A possible function for the CP77 protein during vaccinia virus infectionof CHO cells has been reported by Hsiao et al. (2006). They propose thatCP77 binds to and removes the HMG20A from the newly synthesized vacciniagenome located in the viral factories, thus enabling the vaccinia lifecycle to continue in CHO cells. It is postulated herein that CP77enables the newly synthesized genome to be available for packaging whichotherwise would not be available in CHO cells due to HMG20a binding toand “locking up” the genome. Since the function of CP77 is not requiredfor viral amplification in permissive cell lines, such as Vero, it isproposed herein that there is an alternative equivalent protein, even acellular protein, that has this function which maybe be inactive orabsent in at least CHO cells but active or present in permissive celllines. The alternative protein could render CP77 not required and thusover the evolution of vaccinia it was subsequently deleted orrearranged, as its loss of function was not essential for the broad hostrange.

Example 3 Construction of p-LL07-CHO (Polyclonal Cell Line ExpressingCP77)

A CHO cell line was constructed to express the CP77 protein. A VACV-COPrecombinant virus expressing a green fluorescent protein (EGFP) formsplaques which develop into a confluent infection within a few days ofinfection.

Vaccinia-COP (SCV401C) is a recombinant vaccinia virus of the Copenhagenstrain (VACV-COP) that has inserted into the A39R ORF an expressioncassette consisting of a strong vaccinia virus early/late promoteroperatively linked to the protein coding sequence of Enhanced GreenFluorescent Protein (EGFP) and terminated by the poxvirus earlytranscriptional stop sequence. Upon infection of non-permissive andpermissive cells, EGFP will be expressed within the infected cells whichcan be visualised using a fluorescent microscope. In permissive cells,the green fluorescence can be seen to spread from cell to cell as thevirus spread throughout the population of cell.

The CP77 protein coding sequence was synthetically made by GeneArt GmbH(Germany) by recreating (back translation or reverse translation) theDNA sequence from the amino acid of the CP77 encoded by the 025L ORF ofthe cow poxvirus Brighton Red strain UniProtKB/Swiss-Prot: P12932.1, andcodon optimised for expression in mammalian cell (CHO) cells. See SEQ IDNO: 1 for the codon optimised nucleotide sequence for the expression ofthe protein coding sequence of CP77.

The codon optimized CP77 protein coding sequence from pPH51 (a cloningplasmid harbouring the codon optimized CP77 protein coding sequence) wasPCR amplified using PCR primers where the 5′ primer was designed to adda kozak sequence around the start codon and the 3′ primer was designedto add the flag-Tag sequence prior to the stop codon. The amplified PCRproduct was subcloned into the transposon piggybac vector pJ507-2 (Hyg+)purchased from DNA2.0 Inc (USA) via the Bsa I cloning site. Cloning intothe BsaI effectively replaces the comet GPF coding sequence with theCP77 protein coding sequence to create pLL07. The CP77 now becomes undercontrol of the human constitutive Elongation Factor 1 alpha promoter(EF1a) and is co-expressed with the hygromycin resistance gene once bothhave been stably integrated into the genome of transfected cells.

Transduction of CHO by transposon aided stable insertion of the CP77 andhygromycin resistance expression cassettes: CHO cells were seeded intowells of a 6 well plates so that after an overnight incubation they werearound 50% confluency. Using the Effectene transfection reagent fromQiagen, 1 ug of pLL07 was transfected into 1 well of 50% confluent CHOcells following the manufacturer's instructions. The transfected cellswere then incubated overnight in growth medium. The next day, the mediumwas changed with growth medium containing 500 ug/mL hygromycin B forselecting transduced cells. The selection medium was change every 2-3days and when the transduced cells start to grow in numbers they wererecovered using TrypLE Select (Life Technologies) and seeded into a T25flask for further cell expansion.

Verification of CP77 Expression by Western Blotting (Flag Tagged)

Raising rabbit anti-CP77 sera: Rabbits were injected with a 15 aminoacid peptide linked to KHL protein that represented a short internalamino acid sequence of the CP77 protein. This amino acid sequence was:SGSDVNIRSNNGYTC-amino acid positions 481 to 495 of UniProtKBSwiss-ProtP12932.1 [025LBR CP77 protein] SEQ ID NO: 2.

A total of three injections, 1 month apart, were carried out to raiseantibodies to this KHL conjugated amino acid sequence. Blood from theinjected rabbit were tested by western blot against bacterial expressedand Ni-NTA purified N-terminal His-tagged CP77 protein. The rabbitanti-CP77 serum was shown to clearly recognise recombinant CP77 protein.

Testing for CP77 expression by p-LL07-CHO: Two T25 flasks were seededwith CHO and p-LL07-CHO and cultured until the cell monolayers in eachflask reached 100% confluency. Cells from each flask were harvested,washed with PBS and then resuspended in 200 uL. To this, 50 μl of5×SDS-PAGE loading buffer to each tube of resuspended cells and thenincubated at 98° C. for 5 min. 15 uL of each cell protein extract wasloaded into two 10% SDS-PAGE gels, electrophoresed and then blotted ontoHybond ECL nitrocellulose by electro-blotting.

The electro-blotted membranes were then treated with 5% skimmed milkpowder dissolved in Tris Buffer Saline containing Tween 20 (TBST) for 1hour at room temperature to block all available non-specific antibodybinding sites on the membrane. The membranes were then washed severaltimes in TBST before probing with antibodies. Membrane 1 was probed with1:5000 dilution of anti-DDDDK tag antibody [M2] conjugated in HRP(ab49763, Sapphire Bioscience) overnight at 4° C. Membrane 2 was probedwith 1:100 dilution of anti-CP77 antisera overnight at 4° C., washed 3times with TBST, and then probed with secondary antibody, 1:5000dilution of HRP conjugated anti-rabbit antibody (GE Healthcare) for 2hours. Both membranes were then washed 3 times in TBST and treated withECL western blotting detection reagents (GE Healthcare) and exposed toX-ray film as instructed by the user manual.

Plaque assay in CHO and p-LL7-CHO: CHO and p-LL7-CHO cells were seededinto multiple 6 well plates and were cultured until the cell monolayerswere 100% confluent.

A recombinant Vaccinia virus (Copenhagen strain) harbouring an EGFPexpression cassette (green fluorescent protein) under the control of anearly/late vaccinia virus promoter (SCV401C) was used to infect thecells. Due to the unknown titre of SCV401C, 10 μl of the stock virus wasfirstly diluted in 1 ml of MM medium (Dil 1:1:100 dilution), and theninfected each well with 500 μl of the viral diluent. Day 1 postinfection, high moi infection was noticed where most cells werefluorescing green. It was decided to perform a further 1:20 dilution ofDil 1, by adding 100 μl Dil 1 into 2 ml of MM medium (Dil 2). Then thiswas used to infect each well with 500 μl of Dil 2.

The viral infections were viewed under the fluorescent microscope(Olympus IX51) with GFP filter (Cat #U-MGFPHQ, Olympus). The image wascaptured using celiSens Digital Imaging Software (Olympus).

It was seen from the results that VACV-COP expressing green fluorescentprotein (SCV401C) does not propagate in CHO cells as expected. Thesingle cells fluorescing green are the result of virus entering thecell, expressing its gene including EGFP but unable to produce newinfectious viral particles and therefore unable to spread the infectionto the neighbouring cells. However, in a CHO cell line expressing CP77,the vaccinia virus is able to produce new infectious viruses thatspreading to the neighbouring cells for form a foci of infection by day1 post infection. These foci of infection became a confluent infectionby the next two days post infection where by day 3 the entire cellmonolayer was infected with SCV401C. CHO cells expressing the host rangeprotein, CP77, are permissive to vaccinia virus infections unlike theparental (native) CHO cells.

HMG20A belongs to a family of proteins containing the HMG box domain.HMG proteins are chromosome remodeling proteins that recognize distortedDNA structures, such as cruciforms. They can also induce DNA bending bybinding to the minor groove in DNA. HMG box-containing proteins aretherefore considered important in chromosome remodeling during DNAreplication, recombination, or repair. In addition, certain HMGbox-containing proteins can affect gene transcription by interactingwith transcription factors at the promoter site.

Work published by Hsiao et al. 2006 had shown that CP77 binds HMG20A inCHO-K1 cells. This host cell protein, HMG20A, seems to bind the viralDNA in the viral factories of vaccinia infected CHO cells and it waspostulated that this host cell protein “locks up” the DNA in viralfactories and prevents then next stage of the vaccinia life cycle andthus preventing the production of progeny infectious virus. Expressionof CP77 by the cowpox virus seems to remove host HMG20A off the viralDNA and allow the viral life cycle to recommence with the eventualproduction of progeny infectious virus particles.

However, with the expression of CP77 in CHO in the absence of a viralinfection, one would expect this protein to sequester the newlysynthesized HMG20A present in the cytoplasm before it translocate to thenucleus. If this was the case, the function of the HMG20A in the nucleuswould be lost, and as it plays a critical role during DNA replication,recombination and repair plus its function during gene transcription,one would expect expression of CP77 would harm the integrity of the CHOcell during cell multiplication and maintenance. This unexpectedly doesnot seem to be the case as the CHO cell line expressing CP77 was readilymaintained as a continuous culture with no noticeable effects on itsability to replicate over many generation compared to the parental CHOcell line.

Example 4 Multistep Growth Studies

Multistep growth kinetic study was conducted in p-LL07-CHO: Thepermissive nature of a CHO cell line expressing the host range gene CP77to vaccinia virus infection was assessed and the level of viralproduction to the production levels attained in a naturally permissivehuman cell line—143B was compared.

The aim was to compare the propagation characteristic of VACV-COP inp-LL07-CHO, CHO and 143B cell lines. In this study, the functionality ofthe expressed CP77 by p-LL07-CHO was tested by examining theamplification characteristic of VACV-COP within this cell line comparedto its amplification characteristic in CHO (non-permissive) and 143B(permissive) cells.

Materials and Methods

Cell Line Setup

CHO setup: One 6-Well Plate (6WP) was seeded with CHO cells and wascultured until confluent in growth medium (RPMI+10% FBS).

143B setup: One 6-Well Plate (6WP) was seeded with 143B cells and wascultured until confluent in growth medium (RPMI+10% FBS).

p-LL07-CHO setup: 6-Well Plate (6WP) was seeded with p-LL07-CHO cellsand was cultured until confluent in growth medium (RPMI+10% FBS+500ug/ml HygromycinB).

Dilution of VACV-COP: Each well of each 6WP was infected with 0.01 pfuin a total volume of 500 uL. It was assumed the cell count per well at100% confluency was 4×10⁶cells. For an infection rate of 0.01 pfu/well,4×10⁴ pfu per well was required and so the stock virus was diluted to8×10⁴ pfu/mL in maintenance medium (RPMI/2% FBS).

Infections: Culture medium was removed from each well of each platewhere 500 μL of diluted virus was added and left to incubate at roomtemperature for 1 hr for cell to adsorb the virus. Virus inoculum wasthen removed and each infected well was washed once with 1 mL sterilePBS and then incubated in 2 mL of MM per well at 37° C./5% CO₂.

Harvesting: Two sets of wells from each plate where harvested at thefollowing times post infection: 24 h, 48 h, and 72 h. On the day ofharvesting, cells were scraped into the culture medium where two set ofwells from each cell line was combined and the cells pelleted bycentrifugation at 1000 g for 5 min. Each cell pellet was resuspended in1 mL of 10 mM TrisHCl pH8. The resuspended cell pellet was thensubjected to three cycles of freeze-thawing and stored at −80° C. untilready for titration.

Titrations were done in Vero and 143B cells cultured to 100% confluencyin 24-well plates.

Dilution: The viral extracts were removed from the freezer, thawed andsonicated to homogenise any visible clumps. The viral extracts wereserial diluted to 10⁻⁸ in maintenance medium.

Infections: Growth medium was removed from each well and infected with 1mL of each dilution (4 wells per dilution, one plate for each virusstarting from 10⁻² dilution) for 1 h at room temperature. After theincubation, each plate was move to the incubator and incubated at 37°C./5% CO2 for 3 days for plaques to develop.

Calculation of Titre: the dilution that contains 20 to 50 plaques, theplaques were counted and then averaged. This average count wasmultiplied by the reciprocal of the dilution and because 1 mL infectionwere used, the resulting figure will be the titre in pfu/mL.

Calculation of standard Error: Standard error (SE) at 95% confidence wascalculated from the 4 titration values that constituted the mean usingthe following formula: 1.95×(SD/√n) where: SD is the standard deviationfrom a small sample, n is the number of titration replicates (4 in thiscase).

Calculation of yield: This is the total amount of virus within the viralextract that was being titred: Mean Titration (pfu/mL)×Total volume ofViral Extract=pfu.

Calculation of Amplification Ratio: This figure represents the foldamplification over the amount used as inoculum: Yield in pfu/Inoculumsize in pfu.

Multistep growth kinetic studies were carried out in 6 well plates—twowells per cell line per time point. Each well was infected with 4×10⁴pfu of VACV-COP and for harvesting the 2 well for each cell line andtime point was harvested and combined from which a viral extract wasprepared. This viral extract, total volume of 1 mL represent 2 combinedinfection resulting from an inoculum size of 8×10⁴ pfu (4×10⁴ pfu×2) wasthen titred. Titration was carried out using two indicator cell lines143B and Vero.

The titration and viral yield results are tabulated in Tables 3 and 4respectively. VACV-COP did not amplify in the non-permissive CHO cells,i.e., it produced less virus than the amount used in the input inoculum.However, virus amplification in the CHO cell line expressing thehost-range gene CP77 was about 2000 times more than the input inoculum(based on titration results using 143B cells as the indicator cellline). Amplification from the permissive cell was about 3000 times morethan the input inoculum. Because the standard errors overlap thedifference in amplification between the p-LL07-CHO and 143B cells arenot statistically significant.

If viral amplification between the two cell lines are compared using thetitration results from the Vero indicator cells it would seemamplification was marginally more in the p-LL07-CHO than in the 143Bcells, however this was not statistically significant.

In this study it was further confirmed that vaccinia virus is notpermissive in CHO cells as the level of viral production was very muchless that the amount of virus used as inoculum. However, if CHO expressthe cowpox virus host range gene encoding CP77 protein, it now becomespermissive to vaccinia virus and support the production of virus to thesame levels seen in a permissive cell line. It is also noteworthy thatit only required expression of one host range gene, CP77, to convertthis cell line into a “usable” permissive cell substrate for theproduction of vaccinia. Producing vaccinia from CHO is very desirable asCHO is a biotechnology friendly cell line in that it grows as fast asbacteria, it can be cultured in defined synthetic culture medium withoutthe requirement for biological additives such as Foetal Bovine Serum andcan be cultured as a cell suspension in bioreactor. CHO also has ahistory of producing biological medicinal products, has been wellcharacterised and is known and liked by the biomedical control agenciessuch as FDA and EMEA.

A transgenic CHO cell line expressing the CP77 protein under the controlof a constitutive cellular promoter is permissive to vaccinia virusreplication and propagation to the same levels seen in a naturallypermissive cell line that yields high levels of progeny virus.

Example 5 MVA Propagation in CHO-CP77 Cells MVA+GFP Propagation in CHO,143B and p-LL07-CHO

Materials and Methods

Growth Medium (GM): RPMI-1640, supplemented with 10% FBS, 2 mML-Glutamine, Penicillin and gentamicin, Hepes

Maintenance Medium (MM): RPMI-1640, supplemented with 2% FBS, 2 mML-Glutamine, Penicillin and gentamicin, Hepes

Notes on culturing p-LL07-CHO: General propagation and maintenance ofthe p-LL07-CHO cell line was carried out in GM plus 500 ug/mL ofhygromycin B but however, for plating out and culturing to 100%confluency prior to infection, cells were cultured in GM withouthygromycin B.

Cell setup: 143B, CHO and P-LL07-CHO cells were seeded into multiple6-well plates and were cultured in growth medium (GM) at 37° C./5%CO₂until the cell monolayers were 100% confluent. One plate per cellline was cultured.

Infection

-   -   A recombinant MVA harbouring a GFP expression cassette (green        fluorescent protein) was used to infect cells cultured in the        6-well plates. Due to the unknown titre of MVA-GFP, the virus        was serially diluted in maintenance medium (MM) as follows:        -   Dil 1: 20 ul of the stock virus was diluted in 2 ml of MM            medium (1:100 dilution) and mixed by vigorous vortexing.        -   Dil 2: 500 ul of Dil 1 was added to 4.5 ml of MM (1:10³            dilution) and mixed by vigorous vortexing.        -   Dil 3: 500 ul of Dil 2 was added to 4.5 ml of MM (1:10⁴            dilution) and mixed by vigorous vortexing.        -   Dil 4: 500 ul of Dil 3 was added to 4.5 ml of MM (1:10⁵            dilution) and mixed by vigorous vortexing.    -   One well of each plate was infected with 500 ul of the following        virus dilutions Dil 2, Dil 3, and Dil 4.    -   Each plate was incubated over a 5 day period and examine for the        development and spread of foci of fluorescent cells. In this        study, Dil 4 produced discernable foci of fluorescent cells over        a three day period. The uninfected wells of each plate were        controls for auto-fluorescence.        Microscope Viewing

The viral infection was viewed under the fluorescent microscope (OlympusIX51) with GFP filter (Cat #U-MGFPHQ, Olympus). The image was capturedusing cellSens Digital Imaging Software (Olympus).

Results

The results showed that MVA expressing green fluorescent protein (GFP)does not propagate in CHO and 143B cells as expected. The single cellsfluorescing green are the result of virus entering the cell, expressingit's gene including GFP but unable to produce new infectious viralparticles and therefore unable to spread the infection to theneighbouring cells. However, in a CHO cell line expressing CP77, MVA isable to produce new infectious viruses that spreads to the neighbouringcells to form a foci of infection by day 1 post infection with furtherdevelopment by day 3.

Example 6 Confirmation of Host Range Restriction of MVA Harvested fromp-LL07-CHO Infection

Cell Setup

143B, CHO and BHK-21 cells were seeded into multiple 6-well plates andwere cultured in growth medium (GM) at 37° C./5% CO2 until the cellmonolayers were 100% confluent. One plate per cell line was cultured.

Virus Harvesting from Example 5

The MVA+GFP from Dil 3 infected P-LL07-CHO well in Example 5 washarvested after 5 days of infection as follows:

-   -   Both supernatant and cells were collected from the well and        centrifuge for 5 min at 1000 g to pellet the infected cells.    -   The cell pellet was resuspended in 500 ul of 100 mM Tris-HCl pH8        buffer.    -   The resuspended cell pellet was freeze and thawed at least three        times to release virus from the infected cells.        Infection    -   Due to the unknown titre of the crude viral extract the virus        was serially diluted in MM medium in the same manner as in        Example 5.    -   One well of each plate was infected with 500 μl of the following        virus dilutions Dil 2, Dil 3, and Dil 4.    -   Each plate was incubated over a 5 day period and examine for the        development and spread of foci of fluorescent cells. In this        experiment, Dil 3 produced discernable foci of fluorescent cells        over a three day period.        Microscope Viewing

The viral infection was viewed under the fluorescent microscope (OlympusIX51) with GFP filter (Cat #U-MGFPHQ, Olympus). The image was capturedusing cellSens Digital Imaging Software (Olympus).

Results

MVA that was harvested from the CHO cell line expressing CP77 stillmaintained its restricted host range by not being able to propagate inthe non-permissive cells line CHO (hamster) and 143B cell (human). Thelack for green fluorescent foci over the three day period post infectionof CHO and 143B cells demonstrates that no infectious progeny virus wasproduced in these cell lines. However, this MVA still maintained itshost range for BHK21 cells (hamster) as green fluorescent foci ofinfection could be seen by day 1 post infection which grew in size overthe next 3 days.

Conclusions

-   -   CHO cells expressing the host range protein CP77, are permissive        to MVA infections unlike the parental (native) CHO cells.    -   MVA that was propagated in a CHO cell line expressing CP77 did        not increase its host range to non-permissive cell lines such as        CHO and 143B (human).

Example 7 Construction of pLL07

Background Information: CP77 (CHO codon optimised) CDS PCR product frompPH51 DNA template was cloned into pJ507-2 (Hyg+) PiggyBac system byClontech's InFusion cloning system to create pLL07. The flag-tagged CP77sequence was inserted into the BsaI of pJ507-2 thereby removing thecomet GFP sequence (SEQ ID NO:3).

PCR primer pair used to PCR amplify CP77-CHO gene from pPH51 plasmidDNA:

(SEQ ID NO: 4) AACACGTCTCGGGGGgccgccaccATGTTCGACTACCTGGAAAATGAGGA AGTGand (SEQ ID NO: 5) CAGGAAGACGCTTTTtcaCTTGTCATCGTCATCCTTGTAATCCTGCTGCTCGAAGATCTTGTACT. The Flag Tag sequence is shown in bold in the Inf-LL07-CP77-Rv primer.

The Plasmid INSERT/CASSETTE Configuration is as follows. Insert/CassetteMap. pLL7 clone #3 was sent for sequencing.

15 ABI sequencing files together with the pLL07 reference file“pLL07_ref.sbd” where entered into Lasergene's DNAstar Seqman computerprogram and assembled into 1 consensus contig. The alignment sequenceswhere trimmed to match the start and end of the reference sequencethereafter the reference sequence was deleted from the alignment so ashave no influence on the establishment of the consensus sequence. Theconsensus sequence of the contig was saved as a DNA sequence file named“pLL07_#3 consensus.seq”. The reading direction of the contig wasdetermined and found to represent the same reading direction as thereference sequence. Using Megalign (DNAstar) “pLL07_#3 consensus” wasmanually aligned to “pLL07_ref.sbd” reference sequence to help identifydiscrepancies between the reference sequence and the sequence inpLL07_#3. Assembling the 15AB1 files for pLL07Clone#3 fromAGRF-sequencing service using Seqman (default settings) resulted in oneconsensus contig that covered the full length of the pLL07 insertionreference sequence. There was no discrepancy between the sequence inpLL07_#3 and the reference sequence. The insertion sequences in pLL07_#3contig consensus are identical to the reference sequence.

Example 8 Expression of G1L and I7L in Mammalian Cells

Expression plasmids encoding Flag-tagged-G1L or Flag-tagged-I7L wereconstructed and used to make transgenic 143B cell lines expressing theseproteins. Even though these cells had been amplified in the presence ofGeneticin to positively select transduced cells and PCR analysis hadconfirmed the presence of these protein coding sequences within thegenomes of these cell lines, western blot analysis failed to detect thepresence of expressed Flag-tagged-proteins when probing with ananti-DDDDK antibody.

The protein coding sequences of C-terminal Flag-tagged COP-G1L andCOP-17L where synthesized by GeneArt (Life Technologies) and subclonedinto the Bsa I site of pJ503-2 (piggyBac—neomycin antibiotic selection)purchased from DNA2.0 Inc (USA). This cloning procedure exchanges thecometGFP with the Flag-tagged-G1L or -I7L protein coding sequences. Theresulting clones were designated pLL08 for the G1 L piggyBac vector andpLL10 for the I7L piggyBac vector.

The key features of these two plasmid vectors are: the flag-taggedprotein coding sequences are under the control of constitutive humanpromoter EF 1 alpha, these expression cassette together with the NPT IIexpression cassette (neomycin resistance gene) are flanked by Left andRight Transposon borders to form an artificial transposon element.External to the artificial transposon element but contained within thesame plasmid is the Transposon enzyme expression cassette that mediatesirreversible integration of the transposon element into the host genome.Cells carrying these transposon elements can be positively selected forby including G418 (Geneticin) into the cell growth medium.

The plasmid vector pLL08 (Flag-tagged-G1L) and pLL10 (Flag-tagged-I7L)were transfected into 143B cells to create two transduced transgeniccell lines: G1L-143B (containing the G1L expression cassette), andI7L-143B (containing the I7L expression cassette). Cells with successfultransposon integration were amplified to workable amounts by theinclusion of Geneticin in the growth medium. To verify successfulintegration, total cellular DNA was extracted from these transgeniccells using the DNeasy DNA extraction kit from Qiagen following theinstructions from the kit's instruction manual. Extracted DNA was thenused as template for PCR amplification reactions using PCR primer pairsspecific for PCR amplification of G1L and I7L. Both cell line werepositive for the presence of G1L and I7L DNA sequence in their genomes

To test for G1L and I7L expression by these cell lines Western blotanalysis was carried out by detecting the presence of each Flag-taggedprotein using an anti-Flag Tag antibody [M2] conjugated to HRP(anti-DDDDK antibody, Abcam #ab49763). The results of these western blotanalysis using total protein extracted from the G1L-143B and I7L-143Bcell lines showed that the anti-Flag-tag antibody does not recognise anyproteins extracted from 143B as expected and that if can recognise aFlag-tag protein expressed in a Flag-tag-CP77 transgenic CHO cell line(CP77-CHO) confirming the anti-flag-tag antibody can recogniseflag-tagged proteins. However, no Flag-tagged-G1L protein could bedetected in the protein extract from the G1L-143B sample. Bacteriallyexpressed Flag-tagged-I7L protein can be detected by the anti-Flag-tagantibody but the antibody could not detect Flag-tagged-I7L expression bythe I7L-143B cell line.

Since the G1L and I7L expression cassettes could be detected in theirrespective cell line, the conclusions could be either the expressedproteins are rapidly degraded after synthesis in the absence of avaccinia infection, i.e., both the G1 and I7 proteins are virus specificenzymes and could be unstable without their vaccinia specific enzymesubstrates, or the promoter driving these two expression cassette aredefective. Another hypothesis, expression of these proteins in theabsence of a vaccinia infection are “toxic” to the cells and during theGeneticin selection process, cells that had silenced the transgenic G1Land I7L expression cassettes but not the NPT II expression cassetteenabled the amplification of Geneticin resistant cells that did notexpressed the G1L or I7L proteins. Since pJ503-2 piggyBac plasmid hasbeen used successfully in our lab for the expression of COP-D13L in 143Bcells it is proposed that the EF1alpha promoter was functional and theseproteins were unstable in the absence of a vaccinia infection or thepromoter driving the expression of these proteins where selectivelysilenced to prevent the expression of “toxic” proteins during cellamplification in the presence of Geneticin.

Example 9 D13L As an Example of an Essential Structural Maturation orAssembly Protein is Expressed by Mammalian Cell and Rescues Vacciniawith Deletion of the D13L Gene and D13L Deleted Vaccinia ExpressesProtein in Infected Cells

Construction of D13-Rescue Cell Line—As an example of attenuatingvaccinia by blocking the assembly/maturation process, the D13L ORF ofthe Copenhagen strain was targeted for deletion. In doing so, a cellline expressing this protein would first have to be constructed so thata COP-D13L-deleted virus can be propagated. For construction of therescue cell line the Chinese Hamster Ovary cell line, often referred toas CHO, was chosen as this cell line is “biotechnology” friendly. Inorder to rescue infectious vaccinia virus with a COP-D13L deletion, thiscell line must express the D13-protein from its nuclear genome using thetranscription machinery of the cell and not of vaccinia virus. Theprotein amino acid sequence of the cellular expressed D13-proteincontaining a C-terminal tagged amino acid sequence of DYKDDDDK(Flag-tag, Hopp et al. 1988) and was CHO codon optimized to produce thecorresponding nucleotide sequence. Stable integration of this taggedD13L-CHO codon optimised expression cassette consisting of a mammalianpromoter and a mammalian poly-adenylation signal sequence into thenuclear DNA was achieved by Transposon integration technology of thetype reported by Urschitz et al. 2010 and Matasci et al. 2011.Transduction of CHO was achieved by using the piggy Bac vector systempurchased from DNA2.0 Inc (USA).

Construction of D13L protein coding sequence—The D13L protein codingsequence was synthetically made by GeneArt of LifeTechnologies byrecreating the DNA sequence from the D13-amino acid encoded by the D13LORF of the Vaccinia virus Copenhagen strain and codon optimised forexpression in CHO cells. The protein coding sequence of the VACV-COPD13L ORF is shown in the Sequence listing.

Construction of D13L cell transducing vector—The codon optimizedD13-protein coding sequence (D13LchoTagged) from pLL17 was PCR amplifiedand subcloned into the transposon piggyBac vector pJ503-2 (pHULKpiggyBac Mammalian Expression Vector with CometGFP and Neo+) purchasedfrom DNA2.0 Inc (Cat #pJ503-2) via the Bsa I cloning sites to producepLL19. Cloning between the BsaI by In-Fusion cloning (Clontech: ligasefree cloning) removes the comet GPF coding sequence and replaces it withthe tagged D13-protein coding sequence by in vitro homologousrecombination. The D13LchoTaggedprotein coding sequence is now undercontrol of the human Elongation Factor 1 alpha promoter (EF1a) and willbe co-expressed with the Neomycin resistance gene once both have beenstably integrated into the genome of transfected cells. Stableintegration into the host genome is mediated by Transposon integrationof the DNA sequence bound by the Left and Right Transposon boarder ofthe piggyBac vector.

PCR amplification of the D13LchoTagged sequence was done using thefollowing primer pair:

Forward Primer sequence: Inf-LL19-D13LC-Fw: 5′- AACACGTCTCGGGGGgccgccaccATGAACAACACCATCATCAA-3′

The sequence in uppercase, bold and underline text represent sequencehomologous to the Bsa I site up-stream of the comet GFP in pJ503-2(Neo+) necessary for In-fusion cloning. The sequence in lowercase redand underlined text is a modified Kozak sequence. The sequence in normaluppercase text is homologous to the 5′ end of the D13LchoTagged sequencein pLL17.

Reverse Primer sequence Inf-LL19-D13LC-Rv: 5′- CAGGAAGACGCTTTTTCACTTGTCGTCGTCGTCCTTGTAG-3′

The sequence in uppercase, bold and underline text represent sequencehomologous to the Bsa I site down-stream of the comet GFP in pJ503-2(Neo+) necessary for In-fusion cloning. The sequence in normal uppercasetext is homologous to the 3′ end of the D13LchoTagged sequence in pLL17.The expected 1719 bp PCR product was cloned into BsaI cut pJ503-2 (Neo+)by InFusion cloning (Clontech) following the manufacturer's instructionsto create pLL19.

Construction of a CHO cell line expressing D13 protein: p-LL19-CHO-CHOcells were seeded into wells of a 6-well plate so that after anovernight incubation they were around 50% confluency. Using theEffectene transfection reagent from Qiagen (Cat #301425), 1 ug of pLL19was transfected into 1 well of 50% confluent CHO cells following themanufacturer's instructions. The transfected cells were then incubatedovernight in growth medium (RPMI 1640/10% FBS/2 mM Glutamax/Pen-Strep).The next day, the medium was changed with growth medium containing 1000ug/mL Geneticin for selecting transduced cells. The selection medium waschanged every 2 to 3 days. When the transduced cells grew to over 90 to100% confluency, they were recovered using TrypLE Select(Gibco-Invitrogen Corp, Cat #12563-029) and seeded into a T25 flask forfurther cell expansion.

Verification of D13 expression by Western blotting—Rabbit anti-D13antisera production: Rabbits were injected with a 16 amino acid peptidelinked to KLH protein that represented the C-terminal amino acid of thenative D13-protein. This amino acid sequence was: CYDQGVSITKIMGDNN. Atotal of three injections, 1 month apart, were carried out to raiseantibodies to this amino acid sequence. Serum from the injected rabbitwere tested by western blot analysis against the following cellextracts: 143B whole cell extract, whole extract from 143B transgeniccell line expressing Flag-tagged-D13L (p-LL06-143b) and VACV-COPinfected 143B cell extract. The results clearly demonstrated that therabbit anti-D13 serum can clearly recognise specifically theD13-protein.

CHO-transduced cell preparation—D13LchoTagged transduced CHO polyclonalexpanded cell line (p-LL19-CHO) was cultured to 100% confluency in a T25flask as was normal CHO cells. Cells from each flask were harvested withTrypLE Select (Gibco-Invitrogen Corp, Cat #12563-029), pelleted by lowspeed centrifugation (300 g for 5 minutes) washed with PBS andresuspended in 200 uL of PBS.

Western Blot analysis—50 uL of 5×SDS-PAGE loading buffer was added toeach cell suspension and then incubated at 98° C. for 5 min. 15 uL ofeach cell protein extract was loaded into two 10% SDS-PAGE gels,electrophoresed and then blotted onto Hybond ECL nitrocellulose byelectro-blotting. The electroblotted membranes were then treated with 5%skimmed milk powder dissolved in Tris Buffer Saline containing Tween 20(TBST) for 1 hour at room temperature to block all availablenon-specific antibody binding sites on the membrane. Membranes werewashed several times in TBST before probing with antibodies. Membrane 1was probed with 1:5000 dilution of anti-DDDDK tag antibody [M2]conjugated in HRP (Abcam, Cat #ab49763) overnight at 4° C. Membrane 2was probed with 1:2000 dilution of Rabbit D13-antisera overnight at 4°C., washed 3 times with TBST, and then probed with secondary antibody,1:5000 dilution of HRP conjugated anti-rabbit antibody (Abcam, Cat#ab97069) for 2 hours. Both membranes were then washed 3 times in TBSTand treated with ECL western blotting detection reagents (GE Healthcare)and exposed to X-ray film as instructed by the user manual.

Example 10 Attenuation of VACV-COP by D13L ORF Deletion

To attenuate vaccinia virus, the conserved late promoter sequencetogether with the majority of the protein coding sequence of theCOP-D13L ORF was earmarked for deletion by homologous recombination andin its place, a selection/reporter cassette was inserted so thatsuccessful deletion by homologous recombination can be selected for in aCHO cell line expressing D13-protein and where infection can bevisualised by red fluorescence. The selection/reporter cassette consistof the CHO host range gene expressing CP77 and the DsRed-Express2sequence under the control of a vaccinia promoter.

Construction of COP-D13L deletion homologous recombination vector—Fordeletion of the D13L ORF from VACV-COP by homologous recombination, twohomologous recombination arms were design flanking each side of theCOP-D13L ORF. However, since the promoter sequence of the COP-D12L ORFmight lays within the 3′ end of the COP-D13L ORF, approximately 200 bpof the 3′ end of COP-D13L ORF was left intact.

Construction of homologous recombination arms F1 and F2—The homologousrecombination arms were PCR amplified from VACV-COP genomic DNA usingthe primer pairs shown below. The bold and underline text representIn-Fusion arms for joining the F1 and F2 arms to the selection/reportcassette and the linearized pUC19 supplied in the In-Fusion kit fromClontech. In-Fusion ligation will result in circularized plasmiddesignated pLL09.

PCR Primer Pair Used to PCR Amplify D13L-F1 Arm from VACV-COP DNA:

Inf-PCR-D13L-F1-Fw: 5′ CGGTACCCGGGGATC ACGAAAAATAATAGTAACCA-3′.The bold and underline text (15 bp) represents sequence homologous tothe left end of the linearized pUC19 plasmid supplied by the ClonTechIn-Fusion kit.

Inf-PCR-D13L-F1-Rv: 5′- AATTTAGTGTGCGCG TGGAAAAAGCTTACAATAAACTC-3′.The bold and underline text (15 bp) represents sequence homologous tothe 5′ end of the Selection/Reporter cassette. Expected PCR productsize: 657 bpPCR Primer Pair Used to PCR Amplify D13L-F2 Arm from VACV-COP DNA:

Inf-PCR-D13L-F2-Fw: 5′- ATATTTAAATGCGCG CAATAATGGAACAAGAACCCT-3′.The bold and underline text (15 bp) represents sequence homologous tothe 3′ end of the Selection/Reporter cassette.

Inf-PCR-D13L-F2-Rv: 5′- CGACTCTAGAGGATC GCGCTGAGGTCGGCAACTACG-3′.The bold and underline text (15 bp) represents sequence homologous tothe right end of the linearized pUC19 plasmid supplied by the ClonTechIn-Fusion kit. Expected PCR product size: 621p

Construction of selection/reporter cassette—The selection/reporterexpression cassette consist of the CP77 CHO host-range gene from cowpoxvirus 025L ORF (Brighton Red strain) and the red fluorescent proteincoding sequence of DsRed-Express2. This expression cassette wassynthetically made by Life Technologies GeneArt so that CP77 proteincoding sequence is under the control of its native promoter (100 bp ofsequence upstream of the CP77 ATG start codon) and terminated with thepoxvirus early transcriptional stop sequence (T₅NT). The DsRed proteincoding sequence is under the control of a vaccinia virus early/latepromoter and also terminated in the poxvirus early transcriptional stopsequence.

Assembly of pLL09—The D13L-F1 and D13L-F2 PCR products together with theSelection/Reporter cassette was assembled into pUC19 supplied in theInFusion Kit from ClonTech by In-Fusion cloning following themanufacturer's instructions to produce pLL09.

Deletion of COP-D13L by homologous recombination and plaque purificationto produce SCV104—The COP-D13L ORF was made inactive by deleting themajority of the protein coding sequence and the conserved late promoterelement “TAAAT”. COP-D13L ORF is under the control of a vaccinia viruslate promoter where the conserved late promoter element is critical forpromoter activity (Moss, 2007)—deletion of this will result in theCOP-D13L ORF becoming silenced. Deletion of the protein coding sequenceand conserved promoter element is effected by homologous recombinationbetween VACV-COP and pLL09 where the result of homologous recombinationis the insertion of the CP77/DsRed expression cassette into the deletedregion. Insertion of this expression cassette enables the recombinantvirus, now designated as SCV104, to propagate in CHO cells but onlycells that express a functional D13-protein such as p-LL19-CHO. Afterhomologous recombination contaminating “carryover” parental virus(VACV-COP) was eliminated by successive rounds of plaque purification.The presence of contaminating parental virus was monitored for by PCRanalysis of the insertion site.

Homologous recombination—Three T25 flasks containing growth medium(RPMI-1640/10% FCS/2 mM Glutamax/Pen-Strep and 1000 ug/mL Geneticin)were seeded with p-LL19-CHO and culture until 100% confluent at 37°C./5% CO₂. On the day of infection, two flasks were infected withVACV-COP at an moi 0.01 pfu/cell, where the other flask was not infected(uninfected control). After infecting flask 1 and 2 for 45 min at roomtemperature, the virus inoculums were removed and the monolayer of cellswashed twice with PBS. After washing, 4 ml of Maintenance Medium (MM:RPMI-1640/2% FCS/2 mM Glutamax/Pen-Strep) was added to each flaskincluding flask 3 that had also gone through the same washing step.

Transfection was carried out using Effectene Transfection reagent(Qiagen, Cat No 301425) and following the manufacturer's instructions.Briefly, 16 uL of Enhancer was added to 2 ug of linearized pLL09 in 150uL of EC buffer and left to stand for 5 minutes at room temperatureafter thoroughly mixing. To this 25 μl of Effectene Transfection reagentwas added and left to stand at room temperature for 10 minutes. Finally,1 ml of MM (RPMI-1640/2% FCS/2 mM Glutamax/Pen-Strep) was added to themix and thoroughly mixed gently together. This transfection mix was thenadded to flask 1 that had previously been infected with VACV-COP.

Flasks 1 (homologous recombination), 2 (infection only control), 3(uninfected control) were incubated overnight at 37° C./5% CO₂ where thefollowing day each flask had a media change with fresh MM—5 mL per flaskand further incubated at 37° C./5% CO₂ until gross CPE can be seen inFlask 1 only. There should be no signs of infection in Flask 2 asVACV-COP is non-infectious to CHO cells and the monolayer should lookhealthy in Flask 3.

After the overnight infection red fluorescent cells could be clearlydistinguishable under examination with an inverted fluorescentmicroscope. It was decided to harvest the cells in flask 1 by scrapingthe cells into the culture medium, then pelleted by low speedcentrifugation (500 g for 5 minutes at room temperature) andresuspending the cell pellet in 1 mL of 10 mM Tris-HCl pH8. A viralextract was prepared by multiple freeze and thaw cycles and then storedat −80° C. ready for plaque purification phase. The viral construct wasdesignated SCV 104.

Plaque purification process—The rationale is to serially dilute thehomologous recombination extract and use each dilution to infect one rowof p-LL19-CHO cells cultured in a 48 well plate. The aim is to dilutethe virus down to 1 pfu infection per well and then looking for wellsthat contain only 1 fluorescent plaque after approx. 30 hr of infectionbefore harvesting.

p-LL19-CHO cells were seeded into each well of a 48-well plate andculture to 100% in growth medium (RPMI-1640/10% FBS/2 mMGlutamax/pen-strep) containing 1000 ug/mL Geneticin at 37° C./5% CO₂.

For infection, the homologous recombination extract (SCV104) was thawedand briefly sonicated to break up lumps and aggregates. Ten-fold serialdilution down to 10⁻⁵ of the viral extract was performed using MM(RPMI/2% FBS/Glutamax/PenStrep) in 1 mL volumes. For each dilution, onerow of the 48-well plate was seeded with 500 uL of diluted virus afterremoving the growth medium from each well and washed once with PBS. The48-well plate was left at room temperature for 45 minute for viraladsorption to occur. After viral adsorption, the virus inoculum wascarefully removed from each well where residual inoculum was removed bya washing step consisting of 500 uL of PBS per well. After washing, 500uL of MM (RPMI/2% FBS/Glutamax/PenStrep) was added to each well and thenincubated at 37° C./CO₂ until fluorescent red foci of infections can beclearly seen under a fluorescent microscope.

For harvesting only wells containing a single fluorescent foci at thelowest dilution possible was selected. The medium from selected wellswere carefully removed and 100 uL of 10 mM TrisHCl pH8 was added. Theplate was freeze thawed three times and then the contents of theselected wells were recovered. For each recovered plaque, the plaquepurification process was repeat another five times.

A selected clone was then further amplified by infecting 1 well of a6-well plate containing p-LL19-CHO cells at 100% confluency by removingthe growth medium from the well and adding 10 uL of viral extractdiluted to 500 uL in PBS. After 45 min at room temperature 2 mL of MMwas added to the well and incubated further at 37° C./5% CO₂ for 3 daysuntil majority of the cells fluoresce red under a fluorescentmicroscope. The cells within the infected well were scraped into theculture medium and then pelleted at 500 g for 5 minutes. The pelletedcells were resuspended in 500 uL of 10 mM TrisHCl pH8 and brieflysonicated to make a viral extract.

PCR verification of CP77/DsRed expression cassette insertion into theD13L ORF of SCV104—PCR analysis was carried to determine if afterhomologous recombination and plaque purification that the D13L ORF hadin fact been substituted with the CP77/DsRed expression cassette. A PCRprimer pair was designed to bind outside the region of the two flankinghomologous recombination arms so that they would bind to “native” DNAsequence rather than “introduced” DNA. Designing the primer pair in sucha manner means that this primer pair could use VACV-COP and SCV104 asDNA templates for PCR amplification and the size of the PCR productswill be indicative for insertion of expression cassette into the D13LORF or the detection of the virus with no insertion, i.e., VACV-COP.This PCR assay not only indicates the presence of insertion but also canidentify if residual contaminating parental virus (VACV-COP) is stillpresent after multiple rounds of plaque purification. The presence ofunwanted trace contamination of VACV-COP is highly undesirable as thiscontaminant can provide in trans D13 help to SCV104 and thereby reducethe attenuation of SCV104.

DNA extractions using the QIAGEN DNeasy Tissue Kit (Cat #69504)—ViralDNA was extracted from 200 uL of the above SCV 104 amplified virus usingthe DNeasy Tissue kit following the manufacturer's instruction. Brieflythis was done by adding 20 ul of Proteinase K to 200 uL of viral extractand mixing well. To this, 200 ul of buffer AL was added and thoroughlymixed-in before incubating at 56° C. (heating block) for 10 minutes.After incubation, 200 ul of 100% ethanol was added and mixed-in well andthen the total volume was added to a spin column. The liquid was passedthrough the spin column by centrifugation as instructed by themanufacturer's user handbook followed by spin column washes with AW1 andAW2 buffers. DNA bound to the spin column was eluted off with two time100 uL of AE buffer. The eluted DNA was combined into a single tube andwas ready for PCR analysis or stored at 4° C. until ready for PCRanalysis.

PCR amplification—DNA extracted from SCV104 (see section 4.2.3.1 above)was used a template for PCR amplification to determine if insertion hadtaken place within the D13L ORF. The PCR primers describe below binds tosequences flanking outside the D13L ORF so that amplification fromSCV104 DNA should amplify a PCR product of 4360 bp as appose to a PCRproduct of 2881 bp amplified from the parental virus, VACV-COP. PCRanalysis showed that the 6^(th) plaque purified clones of SCV104 onlyamplify a product corresponding to greater than 4000 bp less than 5000bp indicating SCV104 contains CP77/DsRed cassette in the D13L ORF. Thelack of a PCR product of around 3000 bp means that there are nodetectable levels of parental virus contamination in the SCV 104amplified stocks.

Details of Primer Pair

ID_D12L_LL04_Fw: 5′-TACAAAATCAAATAATGGTCGAAAC-3′ ID_A2L_LL04_Rv:5′-TGCCAAGAAAACACTCCTTCTAAGACAAT-3′

Example 11 Testing SCV104 for Attenuation by Plaque Infectivity Assays

As the protein encoded by the D13L ORF is essential for viral assemblyone would expect that cell entry of an SCV104 rescued virus would not beable to produce infectious progeny virions in normal cells permissivefor vaccinia virus and hence the inability of the initial infection tospread to neighbouring cells to form an ever expanding foci ofinfection. To confirm if this is the case, the D13L deleted virusdescribed in this invention (SCV104) was serially diluted to a pointwhere low number of plaque forming units can be used to infect cellscultured in a 6-well plate so that cell to cell spread of the infectingvirus can be monitored over a period of days. In this study three celllines were studied: 143B cell which are permissive to vaccinia virus,CHO cells which are not permissive to vaccinia virus but can be ifvaccinia virus express CP77 protein and p-LL19-CHO which is arecombinant cell line expressing D13L protein. The virus describe inthis invention which has had a CP77/DsRed expression cassette insertedinto the D13L ORF so that no function D13-protein can be expressedshould only form infectious foci of infection in the p-LL19-CHO cellline. The presence of foci of infections within the 143B cell monolayerindicates the presence of contaminating vaccinia virus within the viralpopulation mix as the contaminant will be providing D13L help in trans.The presence of foci of infections within the CHO monolayer means thatinsertion of CP77/DsRed cassette had not inactivated the D13L ORF.

Cell setup—143B, CHO and p-LL19-CHO cells were seeded into multiple 6well plates and were cultured in growth medium (RPMI-1640/10% FBS/2 mMGlutamax/pen-strep, and only for p-LL19-CHO 1000 ug/mL Geneticin) at 37°C./5% CO₂ until the cell monolayers were 100% confluent.

Infection—Amplified 3r^(d) plaque purified viral extract was used toinfect the cells. Due to the unknown titre of this virus, 10-foldserially diluted of the stock virus was done to 10⁻⁴ as follows:firstly, 60 ul of stock virus was diluted in 6 ml of MM medium (Dil 1;1:100 dilution), and then further dilutions prepared by adding 800 ulDilution 1 to 7.2 ml of MM medium (Dilution 2; 10⁻³ dilution), and thenrepeated to make a 10⁻⁴ dilution. For infection, 500 uL of each dilutionwas added to each well of a 6-well plate—one plate per cell line used.Viral adsorption was carried out at room temperature for 45 min whereafter the viral inoculum was removed from each well and each well washedwith PBS before MM was added at 2 mL per well. The plates were incubatedat 37° C./5% CO₂ and observed daily under a fluorescent microscope. 10⁻²dilution produced the best viral infection rate for observation.

Microscopy viewing—The viral infection was viewed under the fluorescentmicroscope (Olympus IX51) with DsRed filter (Cat #U-MRFPHQ, Olympus).The image was captured using CellSens Digital Imaging Software(Olympus).

Results—10⁻² dilution infection produced the best results for observinginitial single cell infection and the spread of infection toneighbouring cells. Examination of the 143B cell shows the presence ofsingle cell infections at day 1 in which the virus was unable to spreadto neighbouring cells by day 2 and day 3 indicating that the virus entryinto singles cells had occur but no infectious viral progeny had beenproduced by day 2 and day 3. The same is true for the CHO singleinfected cells, i.e., no infectious viral progeny was produced from theinitial single cell infections.

Examination of the p-LL19-CHO cell line shows that the initial singlecell infection had produced progeny infectious virus as seen as smallfoci of infection by day 1. These foci of infections had rapidlyincrease in size by day 2 and day 3 indicating viral amplification istaking place with time.

Accordingly, removal of a functional vaccinia crescent scaffold proteincoding sequence from any vaccinia virus will grossly attenuate thisvirus. Even though this virus is unable to produce infectious progenyvirus upon initial infection, expression of its protein, especially theDsRed, does occur in the initial single infected cells as evidence fromthe visual sight red fluorescence. This virus can be rescued from a cellline expressing the vaccinia crescent scaffold protein independently ofthe vaccinia virus infection. Constitutive expression of the vacciniacrescent scaffold protein by the transgenic cell line has no adverseeffect on the growth or physiology of this cell line.

Example 12 Construction of C11-LL19-HeLa Cell Line Expressing D13L

In order to titrate a non-fluorescent SCV with a D13L deletion, a rescuecell line expressing D13 protein consisting of a vaccinia permissivecell line which supports lytic plaque formation was made. In CHO cells,vaccinia virus expressing CP77 or cell line expression of CP77 does notsupport lytic plaque formations that can be stained with crystal violetand counted by eye. In order to create a titration cell line, HeLa cellswere transduced to express D13 protein by stable integration of anexpression cassette consisting of a constitutive mammalian promoter,D13-protein coding sequence containing the Kozak sequence around thestart codon and terminating in a polyadenylation signal sequence. Thiscassette was then cloned into a plasmid vector that enable stable genetransfer into the host cell genomic DNA and selection for successfulintegration by antibiotic selection. Transduced cells were thenamplified by using antibiotic selection to select for cells containingthe D13 expression cassette and then a plaque assay carried out withSCV104 (D13L ORF deletion) by infecting monolayers of cells at an moi of0.001 pfu per cells and selecting for cell-line clone that produced thelargest plaque size over a 3 day period. SCV104 has had its D13L ORFreplaces with two expression cassette: one for the expression of CP77protein and the other for expression of a red fluorescent protein.Infection of a HeLa cell line expressing D13 protein with SCV104 willresult in red fluorescent lytic plaques.

Construction of pLL19—D13 cell transducing vector: the construction ofpLL19 is described above. In this plasmid the C-terminal Flag-taggedD13-protrein coding sequence is under the control the constitutive humanElongation Factor 1 alpha promoter and stable gene transfer into thecellular genomic DNA is mediated by transposition. Transposition alsoinserts a Neomycin antibiotic selection expression cassette so thattransduced cells can be positively selected for by adding G418/Geneticininto the growth medium.

Stable insertion of the D13-Flag tagged expression cassette into HeLacells by transduction with pLL19: HeLa cells were seeded into a T25flask, and cultured to approximately 50% confluency in RPMI-1640/10%FBS/2 mM Glutamax/pen-strep growth medium. Using the Effectenetransfection reagent from Qiagen (Cat #301425), 1 ug of pLL19 wastransfected into the T25 flask of 50% confluent HeLa cells following themanufacturer's instructions. The transfected cells were then incubatedovernight in growth medium (RPMI 1640/10% FBS/2 mM Glutamax/Pen-Strep).The next day, the medium was changed to fresh growth medium containing1000 ug/mL Geneticin for selecting transduced cells. When most of thecells died the remaining cells were recovered by detaching them from theflask surface with TrypLE Select (Gibco-Invitrogen Corp, Cat #12563-029)and single cell sorted into 96-well plates containing growth medium and1000 ug/mL Geneticin. These plates were incubated at 37° C./5% CO₂ untilcolonies of cells could be seen in each well. The selection medium waschanged every 2 to 3 days. Each colony of cells were recovered andserially expanded by culturing in to the following: 48-well plate to24-well plate to 6-well plate to T75 flask and maintained in T75 flaskby 1:5 split ratio until ready for making a frozen cell stock.

Screening for a monoclonal cell line that best support SCV104 plaqueformation: SCV104 is a vaccinia virus that has had its D13L ORF replacedwith cow poxvirus 025L promoter plus ORF which codes for the CP77protein, and a red fluorescent protein expression cassette (DsRedExpress2). The CP77 expression is not important or needed for plaqueformation in HeLa cells but this virus will not propagated in theabsence of cell line expression of D13 protein. However, in HeLa cellline expressing D13-protein SCV104 should be able to amplify and spreadto other neighbouring cells and in doing so form red fluorescent lyticplaques in the cell monolayer. This virus was used in a plaque formationstudy in clonal LL19-HeLa cell lines to select the clone that bestsupports plaque formation.

Cell line setup: a number of cell line clones of LL19-HeLa were seededinto wells of 6 well plates and cultured in growth medium containing1000 ug/mL Geneticin to 100% confluency at 37° C./5% CO₂:

Virus infection: SCV 104 was used to infect the cells at 0.001 pfu percell by diluting the virus in MM (RPMI-1640/2% FBS/2 mMGlutamax/pen-strep) to 10³ pfu/ml. 1 ml of diluted virus was added toeach well for infection. Each well of a 6-well plate containsapproximately 1×10⁶ cells when confluent, therefore 1 ml of 10³ pfu/mlresults in an moi 0.001. An moi of 0.001 will ensure plaque formationsfrom single infected cells. All plates were incubated at roomtemperature for 1 hour so the virus can adsorb to the cells and thereafter 1 mL of MM was added to each well and all plates were thenincubated at 37° C./5% CO₂ promoting synchronous viral entry into cellsfollowed by viral amplification resulting in cell to cell spread overtime. Red fluorescent plaque formation was observed daily under afluorescent microscope.

Microscopy viewing: viral infection and plaque formation over a threeday period was viewed under the fluorescent microscope (Olympus IX51)with DsRed filter (Cat #U-MRFPHQ, Olympus). The image was captured usingCellSens Digital Imaging Software (Olympus).

Results: A day 1 post infection all infections resulted in sporadicsmall foci of red fluorescence. By day 3, all clonal cell lines producedsizable red fluorescent lytic plaque where clone 11 (C11) produced thesignificantly largest plaque sizes of all clones tested.

Conclusion: These results demonstrate that the C11 clone (C11-LL19-HeLa)was expressing enough D13 protein to support largest plaque formation ofall the clones tested from an SCV104 infection. This cell line clone(C11) is excellent for quantifying a D13L deleted vaccinia virus usinglytic plaque counting method of titration commonly used to titratevaccinia virus.

Example 13 Construction of p-LL07-LL29-CHO Cell Line Expressing CP77andD13L

In order to rescue a VACV-COP virus with a D13L ORF deletion in CHOcells, a CHO cell line expressing the D13 and CP77 proteins would haveto be constructed. This was done by constructing a D13 mammalianexpression cassette and CP77 mammalian expression cassette consisting ofa mammalian promoter to drive expression, a CHO preferred codonoptimised DNA sequencing coding for the D13 or CP77 proteins followed bya polyadenylation signal sequence. These cassettes where then clonedinto plasmid vectors that enable stable gene transfer into the host cellgenomic DNA and selection for successful integration by antibioticselection. Transduced cells were then amplified by using antibioticselection to select for cells that contain both D13 and CP77 expressioncassettes. To verify expression of CP77 a vaccinia virus expressingEnhance Green Fluorescent protein (SCV505) was used to infect thetransgenic cell line where fluorescent plaque development was monitorover a 4 day period. Ever expanding green fluorescent plaque size overthe 4 day period confirmed CP77 expression by the cell line. To verifyexpression of D13 protein and a D13L deleted vaccinia virus expressingCP77 (SCV 104) and DsRed fluorescent protein was used to infect thetransgenic cell line where plaque development was monitor over a 4 dayperiod. Ever expanding red fluorescent plaque size over the 4 day periodconfirmed D13 protein expression by the cell line.

Construction of pLL07—CP77 Gene Transfer Plasmid

Construction of CP77 protein coding sequence: the CP77 protein codingsequence was synthetically made by GeneArt GmbH (Germany) by recreating(back translation or reverse translation) the DNA sequence from theamino acid sequence of the CP77 encoded by the 025L ORF of cow poxvirusBrighton Red strain UniProtKB/Swiss-Prot: P12932.1 and codon optimizedfor expression in Chinese Hamster Ovary (CHO) cells.

Construction of CP77 cell transducing vector: the codon optimized CP77protein coding sequence from pPH51 (a cloning plasmid harbouring thecodon optimized CP77 protein sequence) was PCR amplified using PCRprimer pair where the 5′ primer was designed to add a Kozak sequencearound the start codon and the 3′ primer was designed to add theFlag-tag sequence prior to the stop codon. The amplified PCR product wassubcloned into the transposon piggybac vector pJ507-2 (Hyg⁺) purchasedfrom DNA2.0 Inc (USA) via the BsaI cloning site. Cloning into the BsaIsites effectively replaces the cometGFP coding sequence with the CP77protein coding sequence to create pLL07. The CP77 now becomes under thecontrol of the constitutive human Elongation Factor 1 alpha promoter(EF1a) and is co-expressed with the hygromycin resistance gene once bothhave been stably integrated into the genome of the transfected cells.Stable integration into the host genome is mediated by Transposonintegration of the DNA sequence bound by the Left and Right Transposonboarder of the piggyBac vector.

PCR Primer Pair Used to PCR Amplify CP77-CHO Gene from pPH51 PlasmidDNA:

Forward Primer sequence: (SEQ ID NO: 4)AACACGTCTCGGGGGgccgccaccATGTTCGACTACCTGGAAAATGAGGA AGTGReverse Primer sequence: (SEQ ID NO: 5)CAGGAAGACGCTTTTtcaCTTGTCATCGTCATCCTTGTAATCCTGCTGCT CGAAGATCTTGTACT.The Flag Tag sequence is underlined followed by thestop codon (in lowercase).Construction of pLL29—D13 Gene Transfer Plasmid

The CHO-codon optimized D13 protein coding sequence was PCR amplifiedfrom pLL19 (as described previously) to exchange the C-terminal Flag-tagsequence to the HA-tag sequence before cloning into pJ503-02 (pHULKpiggyBac Mammalian Expression Vector with CometGFP and Neo+) purchasedfrom DNA2.0 Inc (Cat #pJ503-2) via the Bsa I cloning sites.

Construction of D13L-HA cell transducing vector: the codon optimizedD13-protein coding sequence without the C-terminal Flag-tag sequence wasPCR amplified from pLL19 and subcloned into the transposon piggyBacvector pJ503-2 (pHULK piggyBac Mammalian Expression Vector with CometGFPand Neo+) purchased from DNA2.0 Inc (Cat #pJ503-2) via the Bsa I cloningsites to produce pLL29. Cloning between the BsaI sites by In-Fusioncloning (Clontech: ligase free cloning) removes the cometGPF codingsequence and replaces it with the newly HA-tagged D13-protein codingsequence by in vitro homologous recombination. The D13LchoHA-Taggedprotein coding sequence is now under control of the constitutive humanElongation Factor 1 alpha promoter (EF1a) and will be co-expressed withthe Neomycin resistance gene once both have been stably integrated intothe genome of transfected cells. Stable integration into the host genomeis mediated by Transposon integration of the DNA sequence bound by theLeft and Right Transposon boarder of the piggyBac vector. A plasmid mapof pLL29 is shown below.

PCR Amplification of the D13Lcho-HA Tagged Sequence was Done Using theFollowing Primer Pair:

Forward Primer sequence: Inf-LL19-D13LC-Fw:5′-AACACGTCTCGGGGGGCCGCCACCATGAACAACACCATCATCAA-3′Reverse Primer sequence: Inf-LL27-D13L-Rv:5′-CAGGAAGACGCTTTTttaAGCATAATCTGGAACATCATATGGATAGT TGTTATCGCCCATGATCT-3′The sequence in underline text represent HA coding sequence. “tta”represents the stop codon

Stable double insertion of the CP77-Flag tagged and D13-HA taggedexpression cassettes into CHO cells by simultaneous transduction withpLL07 and pLL29

CHO cells were seeded into a T25 flask, and cultured to approximately50% confluency in RPMI-1640/10% FBS/2 mM Glutamax/pen-strep growthmedium. Using the Effectene transfection reagent from Qiagen (Cat#301425), 1 ug of pLL07 and 1 ug of pLL29 were transfected into the T25flask of 50% confluent CHO cells following the manufacturer'sinstructions. The transfected cells were then incubated overnight ingrowth medium (RPMI 1640/10% FBS/2 mM Glutamax/Pen-Strep). The next day,the medium was changed to fresh growth medium containing 500 ug/mLGeneticin and 250 ug/ml Hygromycin B for selecting transduced cells. Theselection medium was changed every 2 to 3 days until most of the cellsdied and detached leaving colonies of cells that were derived fromsingle cells. When the transduced cells grew to over 90 to 100%confluency, they were recovered using TrypLE Select (Gibco-InvitrogenCorp, Cat #12563-029) and seeded into a T75 flask for further cellexpansion. This new polyclonal cell line was designated p-LL07-LL29-CHO.

Verification of D13 and CP77 Expression by Rescuing Vaccinia Virus andVaccinia Virus Deleted of D13L ORF

SCV505 is a vaccinia virus that expresses the Enhanced Green FluorescentProtein (EGFP) and only propagate in CHO cells in the presence of CP77protein. This virus was used in a plaque infectivity study inp-LL07-LL29-CHO to verify CP77 expression by this cell line. SCV104 is avaccinia virus that has had its D13L ORF replace with cow poxvirus 025Lpromoter plus ORF which codes for the CP77 protein, and a redfluorescent protein expression cassette (DsRed Express2). This viruswill not propagated in the absence of cell line expression of D13protein. This virus was used in a plaque infectivity study inp-LL07-LL29-CHO to verify D13 expression by this cell line.

p-LL07-LL29-CHO Cell Line Plaque-Infectivity Study

Various cell lines were used in this study to help qualify theexpression of D13 and CP77 by p-LL07-LL29-CHO from infection by SCV505(D13⁺ CP77⁻ EGFP⁺) and SCV104 (D13⁻ CP77⁺ DsRed⁺). The expected resultsin the following cells lines are as follows:

Vero: This cell line is normally permissive to vaccinia virus infection.Plaque formation indicating cell to cell spread of amplified virus overtime as detected by GREEN fluorescence for SCV505 infection as thisvirus is normally infectious in this cell line. However, no plaqueformation as detected by the lack of or only single cell REDfluorescence for SCV 104 infection should be seen indicating there is nocell to cell spread of amplified virus over time due to the lack of D13protein expression by cell line or virus.

C11-LL19-HeLa: this is a vaccinia virus permissive cell line expressingD13 protein via pLL19 transduction. Plaque formation from SCV505infection is expected indicating cell to cell spread of amplified virusover time as detected by GREEN fluorescence regardless of cell lineexpression of D13. Plaque formation from SCV 104 infection is expectedsince this virus can now amplify in this cell line due to cell lineexpression of D13 protein.

CHO: this cell line is non-permissive to vaccinia virus infection. Noplaque formation from SCV505 and SCV104 infections is expected to beseen as detected by the lack of or only single cell RED or GREENfluorescence. This indicates that there is no cell to cell spread ofamplified virus over time due to the lack of D13 protein expression bycell line needed to rescue SCV104 event though this virus can expressCP77 and the lack of CP77 protein expression by cell line needed torescue SCV505.

p-LL07-LL29-CHO: this is a CHO cell line expressing both D13 and CP77proteins. Plaque formation from SCV505 infection is expected indicatingcell to cell spread of amplified virus over time as detected by GREENfluorescence indicates the cell line expression of CP77 protein. Plaqueformation from SCV104 is expected indicating cell to cell spread ofamplified virus over time as detected by RED fluorescence indicates thecell line expression of D13 protein.

Cell line setups: Vero, CHO, C11-LL19-HeLa, and p-LL07-LL29-CHO celllines were seeded into two sets of multiple 6 well plates (one forSCV505 infection and the other for SCV104 infection) and were culturedin corresponding growth medium (as follows) to 100% confluency at 37°C./5% CO₂:

-   -   Vero, CHO: RPMI-1640/10% FBS/2 mM Glutamax/pen-strep    -   C11-LL19-Hela: RPMI-1640/10% FBS/2 mM Glutamax/pen-strep, plus        1000 ug/mL Geneticin    -   p-LL07-LL29-CHO: RPMI-1640/10% FBS/2 mM Glutamax/pen-strep, plus        500 ug/mL Geneticin and 250 ug/ml Hygromycin B

Virus infection: SCV104 and SCV505 were used to infect the cells at0.001 pfu per cell by diluting the virus in MM (RPMI-1640/2% FBS/2 mMGlutamax/pen-strep) to 10³ pfu/ml. One virus per plate: 1 ml of dilutedvirus was added to each well for infection. Each well of a 6-well platecontains approximately 1×10⁶ cells when confluent, therefore 1 ml of 10³pfu/ml results moi 0.001. At moi of 0.001 will ensure plaque formationsfrom single infected cells. All plates were incubated at roomtemperature for 1 hour so the virus can adsorb to the cells and thereafter 1 mL of MM was added to each well and all plates were thenincubated at 37° C./5% CO₂ promoting synchronous viral entry into cellsfollowed by viral amplification resulting in cell to cell spread overtime. Fluorescent plaque formation was observed daily under afluorescent microscope.

Microscopy viewing: viral infection and plaque formation over a four dayperiod was viewed under the fluorescent microscope (Olympus IX51) withDsRed filter (Cat #U-MRFPHQ, Olympus) for SCV104 virus and GFP filter(Cat #U-MGFPHQ, Olympus) for SCV505. The image was captured usingCellSens Digital Imaging Software (Olympus).

Results:

Infection of CHO cells with SCV104 and SCV505 at moi of 0.001: at day 1post infection only sporadic single cell fluorescence could be seenindicating both virus had entered into the cells but have not yetamplified to spread the infection to the neighbouring cells. However,this remained the same progressing from day 2 to day 4 post infection,i.e., only single cell infections seen and no viral spread toneighbouring cells with time.

Infection of Vero cells with SCV104 and SCV505 at moi of 0.001:infection with SCV505 was as expected, tiny plaques formed at day 1 postinfection which all increased in size to a point where all the plaquesmerged into one confluent infection of the cell monolayers by day 4.This indicated the virus amplified from day 1 onwards to spread theinfection to completeness by day 4 post infection. However, this iscompletely different from the SCV104 infection. At day 1 post infectiononly sporadic single cell fluorescence could be seen which the sameremained over the next 3 days. This indicated that SCV 104 was unableamplify and propagate in this cell line as the virus was lacking theD13L ORF and unable to initiate viral assemble after viral genomereplication and also this cell line did not express D13 protein to helprescue viral amplification.

Infection of C11-LL19-HeLa cells (HeLa cell line expressing D13 protein)with SCV104 and SCV505 at 0.001: infection with SCV505 was as expected,small plaques formed at day 1 post infection which increased in size toa point where all the plaques merged into one confluent infection of thecell monolayers by day 4. This indicated the virus amplified from day 1onwards to spread the infection to completeness by day 4 post infection.Infection with SCV104 also produce the same results demonstrating thatthe D13 protein produced by this cell line complemented for the lack ofthe D13L ORF in SCV104 and the amount produced by this cell line wasenough to support viral amplification and spread of infection comparableto SCV505 which has an intact D13L ORF.

Infection of p-LL07-LL29-CHO cells (CHO cell line expressing D13 andCP77 proteins) with SCV104 and SCV505 at moi 0.001: for both SCV104 andSCV505, tiny foci of infections were observed at day 1 post infection.Over the next 3 days, these foci of infections expanded into everincreasingly larger plaques that eventually merged into confluentinfections by day 4 post infection. This demonstrated that CP77 wasbeing expressed as it supported SCV505 amplification and propagation andD13 protein also expressed as it supported SCV 104 amplification andpropagation.

Conclusion: These results demonstrate that a CHO cell line expressingCP77 and D13 protein can be used as cell substrate for the productionand manufacture of a D13L deleted vaccinia virus. During infection ofnormal permissive cells a D13L deleted virus would not be able toinitiate viral assembly and therefore not complete its infectious lifecycle thus making it an excellent viral vaccine delivery vector forhuman and animal vaccinations in terms of safety. However, this highlyattenuated vaccinia vector can be manufactured in the biotechnologyfriendly CHO cell line if this cell line expresses the CHO host-rangeprotein (CP77) and the missing assembly protein (D13-protein).

Example 14 D13 and CP77 Protein Expression Analysis of p-LL07-LL29-CHOCell Line by Western Blotting

Background Information

The D13 and CP77 proteins expressed by p-LL07-LL29-CHO are tagged andcan be detected using an antibody that specifically recognises the aminoacid tag sequence on the C-terminal ends of D13 and CP77 protein. In theabsence of antibodies that specifically recognise D13 and CP77 proteins,western-blot analysis can be carried out using these anti-tag antibodiesto confirm expression of D13 and CP77 proteins in the p-LL07-LL29-CHOcell line. The C-terminal end of the D13 protein contains the HA-tagamino sequence of “YPYDVPDYA” where the C-terminal end of the CP77protein contains the Flag-tag amino sequence of “DYKDDDDK”.

Western-Blot Analysis Methodology

The following cell lines were seeded into T75 flasks and were culturedto 100% confluency in growth medium (RPMI 1640/10% FBS/2 mMGlutamax/Pen-Strep) containing the appropriate selection antibiotics:CHO without selection antibiotics, p-LL07-LL29-CHO with 500 ug/mLGeneticin and 250 ug/ml Hygromycin B, p-LL07-CHO with 250 ug/mlHygromycin B, and C11-LL19-HeLa with 1000 ug/mL Geneticin. The confluentcell monolayer was detached and digested into single cells suspensionwith TrypLE Select at 37° C. for 10 mins. The cells from each flask wasrecovered and pelleted by centrifugation at 300 g for 5 minutes andwashed twice with PBS. After the final wash the cell pellets wereresuspended in 500 uL of PBS. 4^(x) SDS-PAGE loading buffer was added tothe protein extract to give a final concentration of 1^(x) and heated at98° C. for 2 to 3 minutes. The denatured protein samples wereelectrophoresed through a Biorad Mini-PROTEAN® TGX Stain-Free™ PrecastGel (gradient gel) for 30 to 45 minutes at 200V. After electrophoresis,separated proteins were transferred to nitrocellulose membrane byelectroblotting for 1 hour at 100V.

For protein detection the nitrocellulose membrane was incubated in 5%Skim milk powder in PBS for 1 hour at room temperature to blocknon-specific antibody binding sites. For detection of HA taggedproteins, the membrane was incubated in 1:1000 dilution of Anti-HA HRPconjugated (Abcam Cat #AB1265) in blocking buffer overnight at 4° C. Fordetection of Flag-tagged proteins a separate electroblottednitrocellulose membrane was incubated 1:1000 dilution of Anti-Flag HRPconjugated (Abeam Cat #AB49763) in blocking buffer overnight at 4° C.Membranes were then washed three times in PBS, 5 minutes per wash andthen incubated in ECL substrate (Thermo Scientific Pierce ECLWestern-bot substrate, Cat No 32106) for a couple of minutes beforetaking the imagine using a BioRad XRS gel doc system.

Results

Detection of D13 protein via HA-tag detection: the anti-HA-tag antibodywas able to detect a protein of the expected size from a whole cellprotein extract prepared from p-LL07-LL29-CHO but no protein from awhole cell protein extract prepared from CHO cells. This clearedindicated that p-LL07-LL29-CHO was expressing the D13 protein.

Detection of CP77 protein via Flag-Tag: the anti-Flag-tag antibody wasable to detect a protein of the expected size from a whole cell proteinextract prepared from p-LL07-LL29-CHO and p-LL07-CHO (CHO cell line onlyexpressing CP77) but no protein from a whole cell protein extractprepared from CHO cells. This cleared indicated that p-LL07-LL29-CHO andp-LL07-CHO were expressing the CP77 protein.

Detection of D13 protein via Flag-tag detection: The D13-proteinexpressed by C11-LL19-HeLa expressed the D13-protein with a C-terminalFlag-tag and when a western blot of the whole cell protein extract madefrom this cell line the anti-Flag-tag antibody was able to detect aprotein of the expected size. This cleared indicated that C11-LL19-HeLawas expressing the D13 protein.

Many modifications will be apparent to those skilled in the art withoutdeparting from the scope of the present invention.

TABLE A Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1Nucleotide sequence of CP77 codon optimised for expression in mammaliancells (CHO) 2 Amino acid sequence of CP77 encoded by SEQ ID NO: 1 3Nucleotide sequence of pLL07 4 Forward primer for CP77-CHO gene frompPH51 plasmid 5 Reverse primer for CP77-CHO gene from pPH51 plasmid 6Nucleotide sequence of vaccinia Copenhagen K1L gene (extract fromGenbank M35027.1) 7 Amino acid sequence of vaccinia Copenhagen K1L(extract from Genbank M35027.1) encoded by SEQ ID NO: 6 8VACV-COP-D13L-ORF 9 Nucleotide sequence encoding SEQ ID NO: 1 codonoptimised for hamster cell expression with C-terminal flag-tag (DYKDDDK)10 Forward primer of Inf-LL19-D13LC 11 Reverse primer of Inf-LL19-D13LC12 Flag-tag protein 13 D13L protein peptide 14 Forward primer ofInf-PCR-D13L-F1 15 Reverse primer of Inf-PCR-D13L-F1 16 Forward primerof Inf-PCR-D13L-F2 17 Reverse primer of Inf-PCR-D13L-F2 18 CP77 + DsRedexpression cassette 2903bp 19 CP77 + DsRed expression cassette 20Forward primer of ID_D12L_LL04 21 Reverse primer of ID_A2L_LL04 22 pLL09Homologous recombination vector for the deletion of VACV-COP D13L withCP77/DsRed selection 23 pLL19 Tagged-D13L-CHO codon optimised transposonmediated transducing vector for stable integration into cellular nucleargenomic DNA

TABLE 1 Titration Results 24 h 48 h 72 h CHO 53 pfu/mL 0 0 Vero 8.5 ×10² pfu/mL 14.25 × 10⁶ pfu/mL 27.7 × 10⁶ pfu/mLViral Yields (Output)

The amount of virus used for infection (Input) was 4×10⁴ pfu/mL. Forcomparison sake, yields are expressed as 10⁴ values. Values presentaverage yield per time point, i.e., yield from 3 mL harvest (6 mLdivided by 2 wells).

24 h 48 h 72 h CHO 0.0159 × 10⁴ pfu 0 0 Vero  0.255 × 10⁴ pfu 4275 × 10⁴pfu 8310 × 10⁴ pfuProduction Yield (Output/Input Ratio)

The table below shows the yield of virus produced above the input levelfrom each cell line at each harvest time point, i.e., OUTPUT/INPUTratio.

24 h 48 h 72 h CHO 0.004   0   0 Vero 0.06 1069 2078

TABLE 2 Titration results in pfu/mL Virus/Cell line Dil Count TitreVACV-COP/CHO 10⁻¹ 0 0 pfu/mL 0 0 0 Average 0 VACV-COP/Vero 10⁻⁷ 2  2.8 ×10⁷ pfu/mL +/− 34% 2 4 3 Average 2.8 VACV-PH22/Vero 10⁻⁶ 8 0.75 × 10⁷pfu/MI +/− 43% 5 12 5 Average 7.5 VACV-PH22/CHO 10⁻⁷ 6  7.3 × 10⁷ pfu/mL+/− 20% 9 8 6 Average 7.3Average Virus Output from Infection per Well (Yield)

Virus extract volume per flask was 1 mL. Plating volume for titrationwas 1 mL. Therefore, virus yield equals the titration in pfu/mLmultiplied by 1 mL (plating volume).

Virus Cell line Yield per flask VACV-COP CHO 0 pfu Vero  2.8 × 10⁷ pfuVACV-PH22 Vero 0.75 × 10⁷ pfu CHO  7.3 × 10⁷ pfuYield per pfu Inoculum, i.e., Total pfu Produced from 1 pfu Inoculum

Inoculum size per flask: 1 × 10⁵ pfu Virus Cell line Yield per pfuinoculum VACV- CHO  0 pfu/input pfu COP Vero 280 pfu/input pfu (280 pfuproduced for every pfu used for inoculation) VACV- Vero  75 pfu/inputfpu (75 pfu produced for every PH22 pfu used for inoculation) CHO 730pfu/input pfu (730 pfu produced for every pfu used for inoculation)

TABLE 3 This is the titration of each 1 mL viral extract. Indicator Cellline Cell 143B Vero sub- Titration Titration strate pfu/mL SE % SEpfu/mL SE % SE CHO 5.38E+03 5.74E+02 10.70% 7.25E+02 2.17E+02 30.00% p-1.88E+08 2.45E+07 13.10% 3.95E+07 1.13E+07 28.50% LL07- CHO 143B2.73E+08 3.95E+07 14.50% 3.73E+07 5.45E+06 14.60%

TABLE 4 Table 4 provides the total amount of virus in each 1 mL viralextract. Indicator Cell line 143B Vero Cell Yield Yield substrate pfu SEpfu SE CHO 5.38E+03 5.74E+02 7.25E+02 2.17E+02 p-LL07-CHO 1.88E+082.45E+07 3.95E+07 1.13E+07 143B 2.73E+08 3.95E+07 3.73E+07 5.45E+06

TABLE B Strain ORF Genome Protein Copenhagen COP-D13L M35027 AAA48114Lister clone 107 List-114 DQ121394 ABD52596 LC16mO mO-149L AY678277AAW23819 LC16m8 M8-149L AY678275 AAW23537 WR WR-118 NC_006998 YP_233000Dryvax-3737 VACV-114 DQ377945 ABD57648 Acambis-2000 VACAC2_129 AY313847AAR17961 Acambis Clone 3 VACCL3-129 AY313848 AAQ93215 CVA CVA-124AM501482 CAM58288 Tiantan Clone 10 TT10-148 JX489137 AGJ91839 CowpoxVirus GRI-90 CPXV-GRI-E13L X94355 CAD90667 strain Cowpox Virus BrightonCPXV-131 NC_003663 NP 619914 Red

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The invention claimed is:
 1. A modified Chinese Hamster Ovary (CHO) cellin which the genome of the CHO cell is modified to comprise a sequenceencoding CP77 under the control of a constitutive promoter such that themodified CHO cell line sustains propagation of a vaccinia virus andwherein the genome of the modified CHO cell further comprises a sequenceencoding D13L under the control of a constitutive promoter and/or asequence encoding K1L under the control of a constitutive promoter; withthe proviso that CP77, D13L and K1L do not include functional orthologsand functional variants.
 2. The modified CHO cell of claim 1, whereinthe genome of the modified CHO cell comprises a sequence encoding D13Lunder the control of a constitutive promoter and a sequence encoding K1Lunder the control of a constitutive promoter.
 3. The modified CHO cellof claim 1, wherein the CHO cell is a continuous CHO cell line.
 4. Themodified CHO cell of claim 1, wherein expression of the CP77 genesupports a virus replication amplification ratio of more than
 500. 5.The modified CHO cell of claim 1, wherein the CP77 is encoded by acontiguous sequence of nucleotides codon optimised for expression in CHOcells.
 6. A process for propagating a vaccinia virus in CHO cells, theprocess comprising propagating the vaccinia virus in vitro in a ChineseHamster Ovary (CHO) cell wherein the CHO cell is modified to encode andexpress CP77 under the control of a constitutive promoter and to encodeand express D13L under the control of a constitutive promoter; with theproviso that CP77 and D13L do not include functional orthologs andfunctional variants.
 7. The process of claim 6, wherein the CHO cell ismodified to encode and express K1L under the control of a constitutivepromoter; with the proviso that K1L do not include functional orthologsand functional variants.
 8. The modified CHO cell of claim 1, whereinthe genome of the modified CHO cell comprises a sequence encoding D13Lunder the control of a constitutive promoter.
 9. The modified CHO cellof claim 1, wherein the genome of the modified CHO cell comprises asequence encoding K1L under the control of a constitutive promoter.